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WO2012022741A1 - Circuits synthétiques multicellulaires reprogrammables - Google Patents

Circuits synthétiques multicellulaires reprogrammables Download PDF

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Publication number
WO2012022741A1
WO2012022741A1 PCT/EP2011/064083 EP2011064083W WO2012022741A1 WO 2012022741 A1 WO2012022741 A1 WO 2012022741A1 EP 2011064083 W EP2011064083 W EP 2011064083W WO 2012022741 A1 WO2012022741 A1 WO 2012022741A1
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Prior art keywords
cells
cell
recombinant
different
logic gate
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Ceased
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PCT/EP2011/064083
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English (en)
Inventor
Javier Macia Santamaria
Ricard V. SOLÉ
Francesc Posas Garriga
Eulalia De Nadal Clanchet
Stefan Hohmann
Sergi Regot Rodriguez De Mier
Nuria Conde Pueyo
Kentaro Furukawa
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Institucio Catalana de Recerca i Estudis Avancats ICREA
Universitat Pompeu Fabra UPF
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Institucio Catalana de Recerca i Estudis Avancats ICREA
Universitat Pompeu Fabra UPF
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Publication of WO2012022741A1 publication Critical patent/WO2012022741A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06N—COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
    • G06N3/00—Computing arrangements based on biological models
    • G06N3/002—Biomolecular computers, i.e. using biomolecules, proteins, cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00—Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic

Definitions

  • the invention relates to multicellular engineered systems.
  • Living cells can be seen as computational systems potentially modifiable through appropriate techniques in order to perform arbitrary computations.
  • Examples of synthetic designs include several logic gates, toggle switches, edge detectors, counters, sensors, bistable elements and oscillators. Potential applications span a broad range of areas, from biomedicine to tissue repair or drug synthesis. However, in spite of its great theoretical potential, complex computational constructs (such as comparators, bit adders or multiplexers) are beyond the current state of technology.
  • aspects of the invention relate to the use of combinatorial networks of different cell types to implement complex functions. It has been appreciated that the combination of two or more different cell types, each performing a defined function, can be used to produce a network that performs a more complex function.
  • aspects of the invention can be implemented in a device or system that does not physically separate the different cell types.
  • aspects of the invention may also be implemented by physically separating one or more different cell types, provided that the different cell types can interact (e.g., via a soluble molecule that can be transported and/or diffuse between the different cell types).
  • different types of cells are separated by a permeable or semi-permeable barrier through which soluble molecules can pass.
  • one or more different cells types in a multicellular network may be responsive to one or more non-cellular signals and/or one or more cellular signals.
  • One or more of the different cell types may produce a final output signal (e.g., a detectable signal), wherein the production of the output signal is modulated by at least one cellular signal from one of the other cells in the network.
  • two or more different cell types in a network generate the same cellular signal molecule, but the level of cellular signal is modulated by different input molecules or different combinations of input molecules.
  • the different cell types also are characterized by different functions (e.g., different logic gates) in response to the different input molecules or combinations thereof).
  • aspects of the invention relate to cellular preparations. In some embodiments, aspects of the invention relate to kits.
  • aspects of the invention relate to techniques for performing biological computation by measuring system output(s) as a function of system input(s). Accordingly, aspects of the invention may include methods and devices for containing multicellular systems and providing inputs and reading outputs.
  • complex functions of the invention may be implemented to perform defined tasks in agricultural, industrial, and/or medical contexts.
  • compositions including two or more different types of recombinant cells, wherein each type of recombinant cell responds to a different input signal or combination of input signals, and/or wherein each type of recombinant cell comprises a different logic gate selected from the group consisting of IDENTITY, AND, OR, NOT, NAND, NOR, XOR, XNOR, IMPLIES and N-IMPLIES.
  • at least two of the different types of recombinant cells produce the same output signal.
  • at least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N-EVIPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells. In some embodiments, the recombinant cells are prokaryotic cells such as bacterial cells. In some embodiments, the composition comprises cells of different species or genera. In some embodiments, each cell type is of a different species or genus.
  • compositions including two or more different types of recombinant cells, wherein each type of recombinant cell responds to a different input signal or combination of input signals, and/or wherein each type of recombinant cell comprises a different logic gate selected from the group consisting of IDENTITY, AND, OR, NOT, NAND, NOR, XOR, XNOR, IMPLIES and N-IMPLIES, and wherein two or more subgroups of the different types of recombinant cells are spatially separated from each other.
  • At least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N-IMPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells.
  • the recombinant cells are prokaryotic cells such as bacterial cells.
  • the composition comprises cells of different species or genera.
  • each cell type is of a different species or genus.
  • the two or more subgroups of the different types of recombinant cells are spatially separated within a microfluidic device.
  • each type of recombinant cell responds to a different input signal or combination of input signals.
  • each type of recombinant cell comprises a different logic gate selected from the group consisting of IDENTITY, AND, OR, NOT, NAND, NOR, XOR, XNOR, IMPLIES and N-IMPLIES.
  • the presence or absence of an output signal is detected or measured.
  • at least two different types of the recombinant cells produce the same output signal.
  • At least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N- EVIPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells.
  • the recombinant cells are prokaryotic cells such as bacterial cells.
  • the composition comprises cells of different species or genera. In some embodiments, each cell type is of a different species or genus.
  • aspects of the invention relate to methods including providing one or more input signals to two or more different types of recombinant cells, wherein each type of recombinant cell responds to a different input signal or combination of input signals, and/or wherein each type of recombinant cell comprises a different logic gate selected from the group consisting of IDENTITY, AND, OR, NOT, NAND, NOR, XOR, XNOR, IMPLIES and N-IMPLIES, and
  • At least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N-IMPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells.
  • the recombinant cells are prokaryotic cells such as bacterial cells.
  • the composition comprises cells of different species or genera.
  • each cell type is of a different species or genus.
  • the two or more subgroups of the different types of recombinant cells are spatially separated within a microfluidic device.
  • aspects of the invention relate to methods for conducting biological computation, including providing one or more input signals to a composition comprising two or more different types of recombinant cells, wherein each type of recombinant cell responds to a different input signal or combination of input signals, and/or wherein each type of recombinant cell comprises a different logic gate selected from the group consisting of IDENTITY, AND, OR, NOT, NAND, NOR, XOR, XNOR, IMPLIES and N-IMPLIES, and measuring the presence or absence of an output signal.
  • at least two different types of the recombinant cells produce the same output signal.
  • the output signal is converted into a digital signal.
  • At least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N-IMPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells.
  • the recombinant cells are prokaryotic cells such as bacterial cells.
  • the composition comprises cells of different species or genera. In some embodiments, each cell type is of a different species or genus.
  • kits including at least one pair of recombinant cells
  • each pair of recombinant cells responds to one or more different input signals, wherein within each pair of recombinant cells, one cell comprises an IDENTITY logic gate and the other cell comprises an INVERTER (NOT) logic gate.
  • each pair of recombinant cells produces the same output signal.
  • the kits comprises a recombinant reporter cell that responds to the diffusible molecule and produces a detectable output signal.
  • At least one type of recombinant cell comprises an AND logic gate, an IMPLIES logic gate, an N-IMPLIES logic gate and/or a NOT logic gate.
  • the recombinant cells are eukaryotic cells such as yeast cells or mammalian cells.
  • the recombinant cells are prokaryotic cells such as bacterial cells.
  • the composition comprises cells of different species or genera. In some embodiments, each cell type is of a different species or genus.
  • aspects of the invention relate to one or more recombinant cells described herein.
  • input signals e.g., one or more, for example, 2, 3, 4, 5, or more external signals and/or one or more, for example 2, 3, 4, 5, or more signals produced by another cell.
  • multicellular systems described herein involve wiring, while in other embodiments, multicellular systems described herein do not involve wiring. Some multicellular systems described herein include wiring and wireless components.
  • recombinant cells associated with the invention produce and/or respond to one or more wires.
  • FIG. 1 illustrates non-limiting embodiments of distributed cellular networks
  • FIG. 1 A illustrates a network in which the output of a first cell is modulated by a signal from a second cell
  • FIG. IB illustrates the network of 1 A in which the output of the first cell also is modulated by a first external signal
  • FIG. 1C illustrates the network of 1A in which the output of the first cell is modulated by signals from both the second cell and a third cell
  • FIG. ID illustrates the network of 1C in which the output of the first cell also is modulated by a first external signal, and the signal of the second cell is modulated both by a signal from a third cell and by a second external signal
  • FIG. 1 A illustrates a network in which the output of a first cell is modulated by a signal from a second cell
  • FIG. IB illustrates the network of 1 A in which the output of the first cell also is modulated by a first external signal
  • FIG. 1C illustrates the network of 1A in which the output of the
  • FIG. 2 illustrates a non-limiting schematic of engineered cells
  • FIG. 2A illustrates a cell which receives a signal from another cell (IN) and a signal from an external source (E);
  • FIG. 2B illustrates a cell that just receives a signal from an external source - both cells produce a diffusive output molecule;
  • FIG. 2C presents a schematic representation of cell behavior, where each cell Cy responds to two different inputs namely an external input x, and an internal wire ⁇ 3 ⁇ 4. ⁇ ;
  • FIG. 2D and FIG. 2E illustrate four representative functions that can be implemented in multicellular networks described herein);
  • FIG. 3 illustrates a non-limiting embodiment of a general architecture of multicellular circuits with a fixed wiring pattern
  • FIG. 4 illustrates examples of cells engineered to implement different logic gates in vivo
  • FIG. 4A presents an example of a cellular AND gate and corresponding truth table
  • FIG. 4B presents an example of a cellular NOR gate and corresponding truth table
  • FIG. 4C presents an example of a cellular OR gate and corresponding truth table
  • FIG. 4D presents an example of a cellular NAND gate and corresponding truth table
  • FIG. 5 illustrates a non-limiting design and in vivo implementation of a multiplexer (MUX2al) and 1-bit adder with carry
  • FIG. 5A presents a schematic diagram of a putative transcription based single cell MUX2al
  • FIG. 5B presents a schematic of in vivo implementation of the MUX2tol circuit via distributed computation in engineered cells
  • FIG. 5C demonstrates in vivo implementation of the 1-bit adder with carry
  • FIG. 6 illustrates a non-limiting method for designing distributed biological computation
  • FIG. 6A presents a truth table defining the behavior of a multiplexer circuit
  • FIG. 6B presents a full Boolean function that expresses the relationship between the logic inputs xl, x2, x3 and the logic output O according to the truth table
  • FIG. 6C presents a simplified version of the Boolean function / describing the same truth table
  • FIG. 6D demonstrates implementation of the circuit with direct mapping between the terms of the Boolean function and the engineered cells
  • FIG. 6E demonstrates a mixed implementation, where the set pi is a direct mapping of the terms from the Boolean function whereas in p2 all the terms have been condensed in a single cell
  • FIG. 6F demonstrates circuit implementation where both sets pi and p2 are implemented by a single cell in each case);
  • FIG. 7 presents non-limiting graphs demonstrating the number of possible Boolean functions versus the number of different cells required for their implementation (each graph represent the number of (non-null) functions that can be implemented with a defined number of engineered cells that receive 2-inputs illustrated in FIG. 7A and 3- inputs illustrated in FIG. 7B);
  • FIG. 8 illustrates non-limiting examples of the implementation of N-FMPLIES and IMPLIES circuits
  • FIG. 8A presents a schematic representation of a cell with an N- IMPLIES logic and the corresponding truth table
  • FIG. 8B presents a schematic representation of N-FMPLIES logic implemented in two cells with different logic
  • FIG. 8C presents a schematic demonstration of cells described in FIG. 4C used to implement an IMPLIES logic circuit
  • FIG. 9 illustrates non-limiting examples of circuit reprogramming and associated data
  • FIG. 9A presents AND gate reprogramming including a schematic representation of cells used in the AND circuit, and a corresponding truth table with a third input to reprogram the circuit
  • FIG. 9B presents OR gate reprogramming including a schematic representation of cells and a corresponding truth table
  • FIG. 10 illustrates a schematic summary of nine non-limiting examples of logic gates implemented by single or multicellular components
  • FIG. 11 illustrates a spatially distributed, multicellular implementation of a circuit
  • FIG. 11A depicts a schematic of a non-limiting example of a spatially distributed, multicellular circuit
  • FIG. 1 IB depicts a schematic representation of a CellASIC microfluidic plate, Hayward, CA);
  • FIG. 12 provides non-limiting schematic representations of the biological implementation of two fundamental circuits in electronics (FIG. 12A represents a multiplexor; FIG. 12B represents a comparator; and FIG. 12C depicts a truth table corresponding to the multiplexor in FIG. 12 A);
  • FIG. 13 provides a non-limiting schematic representation of the re- programmability of the cellular circuits described herein (FIG. 13 A illustrates a microfluidic implementation of a function described by the corresponding truth table; and FIG. 13B shows the implementation of a different function and the corresponding truth table);
  • FIG. 14 illustrates a non-limiting example of a library of engineered cells and a universal reporter strain
  • FIG. 15 provides a non-limiting graphical demonstration of a reporter readout by FACS (in FIG. 15A and FIG. 15B, yeast strains carrying the reporter GALl .GVP were grown with glucose (repressed conditions) or galactose (activated conditions); and in FIG. 15C and FIG. 15D, a Jet-ON: :GFP reporter was activated by adding doxicycline);
  • FIG. 16 provides a schematic representation of self-configurable systems (FIG.
  • FIG. 16A depicts a system for functions with one-input and one-output; and FIG. 16B depicts an extension of the self-configurable system for functions with multiple inputs);
  • FIG. 17 depicts a basic circuit in mammalian cells with an AND gate
  • FIG. 18 depicts an embodiment of a circuit for regulating glucose levels in yeast
  • FIG. 19 depicts an example of a multicellular circuit. All cells involved in the control circuit secrete N-3-(oxohexanoly)-HSL synthesized by Luxl and detected by LuxR. Cell #1 responds at low levels of N-3-(oxohexanoly)-HSL whereas Cell #2 has a mutated form of LuxR*, which is activated at high levels of N-3-(oxohexanoly)-HSL. Without pathogenic AHL, Cell#2 secretes N-3-(oxododecanioil)-L-HSL, synthesized by Lasl.
  • FIG. 20 depicts in vivo analyses of engineered cells and transfer functions of basic logic cells
  • FIG. 20A demonstrates quantification of single cell computational output, a truth table corresponding to NOT logic function and a schematic representation of a cell with a NOT logic.
  • the NOT function is implemented in Cell#3, and the reporter cell (Cell#6) is used to quantify alpha factor production in vivo
  • FIG. 20B depicts schematic representations of cells implementing N-IMPLIES, AND, IDENTITY and NOT functions);
  • FIG. 21 depicts design and in vivo implementation of a multiplexer (MUX2tol) and 1-bit adder with carry
  • FIG. 21A demonstrates a schematic representation of cells used in a MUX2t01 circuit with two wiring molecules and a corresponding truth table
  • FIG. 2 IB demonstrates a schematic representation of cells used in a 1-bit adder with carry and a corresponding truth table
  • FIG. 22 depicts mathematical analysis of possible 3-input 1 -output Boolean functions versus the number of different wires required for their implementation upon different approaches (FIG. 22A demonstrates a multicellular approach; FIG. 22B demonstrates a standard approach based on NAND logic; and FIG. 22C demonstrates a standard approach based on NOR logic);
  • FIG. 23 depicts a non-limiting example of an engineered yeast cell library
  • FIG. 23A demonstrates a schematic representation of cells that express a wiring molecule (left) and sensor cells (right); and
  • FIG. 23B demonstrates schematic representations of cells within the library indicating the logic function performed by each cell. Some cells can implement different functions depending on the experimental conditions and the input used (in brackets));
  • FIG. 24 depicts transfer function analyses of engineered cells of the library. Data are presented as a regular graph for 1 input analyses and as a contour plot for 2 input analyses);
  • FIG. 25 depicts logic gates implemented in vivo using glucose and doxycycline as inputs
  • FIG. 25A demonstrates a schematic representation of cells used to implement an AND gate with glucose and doxycycline as inputs
  • FIG. 25B demonstrates a schematic representation of cells used to implement an NOR gate
  • FIG 25C demonstrates a schematic representation of cells used to implement an OR gate
  • FIG 25D demonstrates a schematic representation of cells used to implement an NAND gate
  • FIG 25E demonstrates a schematic representation of cells used to implement an XNOR gate
  • FIG 25F demonstrates a schematic representation of cells used to implement an XOR gate
  • FIG. 26 depicts in vivo characterization of the properties of an AND gate (FIG. 26A demonstrates output duration upon input addition; FIG. 26B demonstrates stability of the circuit over time; and FIG. 26C demonstrates dynamic response of a circuit using a microfluidics platform);
  • FIG. 27 depicts a schematic representation of cells used to implement indicated logic gates, corresponding truth tables and FACS plots for each condition in the truth table (FIG. 27A demonstrates an AND gate; and FIG. 27B demonstrates an NAND gate);
  • FIG. 28 depicts schematic representations of cells used to implement indicated logic gates, corresponding truth tables and FACS plots for each condition in the truth table (FIG. 28A demonstrates an OR logic gate; and FIG. 28B demonstrates an NOR gate);
  • FIG. 29 depicts a schematic representation of cells used to implement a 3 cell- based MUX2tol circuit, a corresponding truth table and a FACS plot for each condition in the truth table;
  • FIG. 30 depicts schematic representations of cells used to implement indicated logic gates, corresponding truth tables and FACS plots for each condition in the truth table (FIG. 30A demonstrates an AND logic gate; and FIG. 30B demonstrates an NAND gate);
  • FIG. 31 depicts schematic representations of cells used to implement indicated logic gates, corresponding truth tables and FACS plots for each condition in the truth table (FIG. 31A demonstrates an OR logic gate; and FIG. 3 IB demonstrates an NOR gate);
  • FIG. 32 depicts schematic representations of cells used to implement indicated logic gates, corresponding truth tables and FACS plots for each condition in the truth table (FIG. 32A demonstrates an XOR logic gate; and FIG. 32B demonstrates an XNOR gate);
  • FIG. 33 depicts a schematic representation of cells used to implement a 4 cell- based MUX2tol circuit, a corresponding truth table and a FACS plot for each condition in the truth table;
  • FIG. 34 depicts a schematic representation of cells used to implement a 1 bit adder with carry circuit, a corresponding truth table and a FACS plot for each condition in the truth table.
  • Each density plot displays GFP and mCherry fluorescent intensity;
  • FIG. 35 depicts FACS plots of controls used in a cellular implementation of logic gates (FIG. 35 A corresponds to an AND logic gate; and FIG. 35B corresponds to an NAND logic gate);
  • FIG. 36 depicts FACS plots of controls used in a cellular implementation of logic gates (FIG. 36A corresponds to an OR logic gate; and FIG. 36B corresponds to an NOR logic gate);
  • FIG. 37 depicts FACS plots of controls used in a cellular implementation of a 3 cell-based MUX2tol circuit
  • FIG. 38 depicts FACS plots of controls used in a cellular implementation of logic gates (FIG. 38A corresponds to an AND logic gate; and FIG. 38B corresponds to an NAND logic gate);
  • FIG. 39 depicts FACS plots of controls used in a cellular implementation of logic gates (FIG. 39A corresponds to an OR logic gate; and FIG. 39B corresponds to an NOR logic gate);
  • FIG. 40 depicts FACS plots of controls used in a cellular implementation of logic gates (FIG. 40A corresponds to an XOR logic gate; and FIG. 40B corresponds to an XNOR logic gate);
  • FIG. 41 depicts FACS plots of controls used in a cellular implementation of a 4 cell-based MUX2tol circuit.
  • FIG. 42 depicts FACS plots of controls used in a cellular implementation of a 1 bit adder with carry circuit.
  • FIG. 43 depicts a one bit memory device (FIG. 43A presents a schematic of a memory device and a corresponding truth table; and FIG. 43B presents a schematic of a signal restoration circuit);
  • FIG. 44 depicts multicellular systems using inverted logic (FIG. 44A depicts a general pattern of connections for an arbitrary Boolean circuit;
  • FIG. 44B depicts an example of the minimal pattern of connections obtained by an evolutive algorithm);
  • FIG. 45 illustrates spatial separation of multicellular circuits (e.g., in microfluidics devices)
  • FIG. 45A depicts a schematic of spatial separation of cells within such a system
  • FIG 45B demonstrates that spatial separation allows for simplification of circuit implementation
  • FIG. 45C demonstrates a correlation between the maximum number of required chambers (line 1) and the maximum number of different functions that can be implemented (line 2));
  • FIG. 46 depicts a schematic diagram of re-programmability (FIG. 46A illustrates an XOR function and corresponding truth table; FIG. 46B illustrates an IMPLIES function and corresponding truth table);
  • FIG. 47 depicts an example of a general SYNCOM chip for function with three inputs (FIG. 47A shows an example where upon inhibition, the corresponding cells are not able to produce the diffusible molecule acting as a wire; FIG. 47B shows an example whereby introducing a specific combination of inhibitors, mixed with the inputs, causes the circuit to be reprogrammed to implement a particular Boolean function; FIG. 47C shows that the same device can be reprogrammed to implement a different Boolean function);
  • FIG. 48 depicts a distributed computation circuit without extracellular wiring
  • FIG. 48A shows a schematic diagram of the general architecture of distributed computation circuits using isolated cells without extracellular wiring
  • FIG. 48B shows a schematic representation of the implementation of the circuit shown in FIG. 44B). Arrows indicate activator inputs, whereas line-dot represents inhibitory inputs.
  • aspects of the invention provide a logically different cellular system for performing computations by combining different cellular units, each having a defined function, in order to produce multicellular systems that implement more complex functions.
  • a simple multicellular system comprises a first cell type that generates an output (e.g., a detectable output), the production of which can be modulated by a signal from a second cell type as illustrated, for example, in FIG. 1A.
  • modulation can refer to increasing or decreasing.
  • the signal from the second cell type can be a diffusible molecule.
  • a signal molecule may be bound to the second cell type and the signal may be mediated by direct cell to cell contact between the first and second cell types.
  • an external signal molecule also may modulate the production of the output by the first cell as illustrated, for example, in FIG. IB.
  • an external signal molecule refers to a signal molecule that is not produced by one of the recombinant cells within a multicellular system described herein.
  • An external signal molecule can be a cellular-generated signal or a non cellular- generated signal but is external to the multicellular system.
  • one or more cells in a multicellular system that respond to an external signal are referred to as "sensor" cells.
  • a signal from an additional cell type also may modulate the production of the output.
  • two or more non-cellular signal molecules and/or two or more cellular signals all may interact with the first cell and modulate production of the output.
  • the level of output will depend on the cumulative effect of all of the signals on the genes and/or proteins that produce the output molecule in the first cell type.
  • the first cell type may be engineered to implement a specific function (e.g., a specific logic function) in response to the one or more input signals.
  • each cell in a multicellular system may be modulated by one or more cellular and/or non-cellular signals.
  • ID illustrates a non-limiting example of a multicellular network wherein production of the output signal is modulated by a combination of two cellular signals (from the second and third cell types illustrated in FIG. ID) and one non-cellular signal, and wherein the cellular signals from the second cell type itself is modulated by cellular signal from another cell (the fourth cell type illustrated in FIG. ID) and by a second non-cellular signal.
  • the different cell types may represent different functions (e.g., different logic functions).
  • the signals generated by each cell type may be different. However, in some embodiments, two or more different cell types may produce the same signal molecule.
  • multicellular systems described herein can be designed and/or engineered to implement complex functions based on a small number of different cell types each implementing a simpler function. It should be appreciated that in some embodiments, each different cell type may represent a different logic function. However, in some embodiments, two or more different cell types may represent the same logic function, but differ in the identity of one or more input and/or output molecules. In some embodiments, aspects of the invention can avoid wiring constraints through the use of a redundant distribution of the desired output among different cellular units.
  • aspects of the invention can be implemented in any type of cell.
  • aspects of the invention can be implemented through the development of a library of engineered cells, which can be combined in multiple ways. The combining of such cells allows for the building of complex synthetic systems and devices.
  • logic functions can be implemented by combining just a few engineered cells.
  • small modifications and combinations of cells allows for complex circuits to be implemented.
  • complex circuits such as a multiplexer or a 1-bit adder with carry can be implemented by combining a few different cell types.
  • other complex circuits can similarly be implemented by combining a few different cell types.
  • circuits include multiplexers, comparators, adders and flip-flops. It should be appreciated that any type of combinatorial or sequential circuit could be implemented according to aspects of the invention.
  • aspects of the invention allow for the re-utilization of components of multicellular circuits. In some embodiments, this allows for systems to be reprogrammed by adding, removing, and/or substituting different cell types in a multi-cellular network.
  • aspects of the invention are not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
  • recombinant cell refers to a cell that has been genetically engineered.
  • different types of recombinant cells refers to more than one type of cell, wherein each type of cell has been engineered differently. It should be appreciated that different types of recombinant cells may respond to the same input or inputs but may respond in a different manner.
  • a sufficient number of individual cells may be provided to perform the desired function (e.g., a particular logic function in response to one or more input signals that generates an output signal that can act as a detectable output or as a signal for a different cell type).
  • the desired function e.g., a particular logic function in response to one or more input signals that generates an output signal that can act as a detectable output or as a signal for a different cell type.
  • the number of cells of a particular type required to perform a desired function will depend on the function and the level of expression of a recombinant gene in each cell.
  • any number of individual cells of each cell type e.g., from about 1,000 to about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or more, for example about 10 15 ), or more or less depending on the identity of the input and/or output and/or the computation being performed. It should be appreciated that different cell types may be provided in different amounts depending on the functions performed by each cell type and/or the properties of each cell type.
  • multicellular molecular circuit As used herein, “multicellular molecular circuit,” “multicellular molecular system,” and “multicellular system” are used interchangeably and refer to a composition comprising two or more recombinant cells that interact with each other and perform a function.
  • the function corresponds to a logic function.
  • the multicellular molecular circuit can be described by a Boolean function.
  • a Boolean function is a function whose range is ⁇ 0,1 ⁇ .
  • the multicellular molecular circuits described herein exhibit distributed computation.
  • distributed computation within a multicellular molecular circuit refers to a system wherein each cell within the multicellular circuit represents a function and the combination of the two or more cells within the multicellular circuit represents a different function than that of either cell individually.
  • the multicellular molecular circuits described herein comprise 2 or more different cell types.
  • the multicellular molecular circuits comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 different cell types and can comprise any number of cells representing each cell type. It should be appreciated that the number of cells of each cell type used in a multicellular network may be optimized depending on the application and the scale of the network.
  • Recombinant cells associated with the invention detect and respond to one or more input signals.
  • an input signal can be any signal to which a cell is capable of responding. The input signal will vary depending on the cell type. In some embodiments, an input signal is a diffusible molecule. An input signal can be an organic molecule or an inorganic molecule.
  • Non-limiting examples of molecules that can serve as input signals in multicellular circuits include pheromones such as yeast pheromones including, for example, Sc alpha-factor or Ca alpha factor, biomolecules such as lactones, phosphoserine or cytokines, small molecules such as doxicycline or methionine, heavy metals such as copper, sugars such as glucose or galactose, and chemical inhibitors such as 1NMPP1.
  • an input signal can be a physical signal that is not mediated by a specific molecule, for example, temperature (e.g., an increase or decrease in temperature), radiation (e.g., photons or gamma radiation), electromagnetic radiation such as ultraviolet radiation, magnetic force or electrical stimulation.
  • Recombinant cells associated with the invention produce output molecules.
  • an output molecule can be any signal produced by a cell. The output signal will vary depending on the cell type. In some embodiments, an output signal is a diffusible molecule. An output signal can be an organic molecule or an inorganic molecule. In some embodiments, an output signal is detectable.
  • Non- limiting examples of molecules that can serve as output signals in multicellular circuits include molecules that can be synthesized and/or metabolized by a cell or cellular component. Several non-limiting examples of output molecules include GFP, CFP, mCherry and YFP.
  • An output molecule can serve as a signal that is transmitted between cells, such that the output molecule of one cell corresponds to an input signal for another cell within the multicellular molecular circuit.
  • a signal that is transmitted between cells is referred to herein as a "wire” and the molecular interconnection between recombinant cells within the multicellular molecular circuits described herein is referred to as "wiring.”
  • a wire is bound to the surface of the cell, while in other embodiments, a wire is diffusible.
  • multicellular systems described herein can have varying numbers of wires.
  • the multicellular systems have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 wires.
  • an initial increase in the number of wires within a multicellular system can cause a large increase in functions that can be performed by the system.
  • a wire can be any molecule or signal that is transmitted between cells.
  • a wire is a diffusible molecule such as a pheromone.
  • aspects of the invention include embodiments wherein the multicellular circuit does not require wiring between cells.
  • Example 11 and FIG. 48 describe distributed computation circuits that use isolated cells to generate multicellular circuits that do not require extracellular wiring.
  • some cells within a multicellular circuit comprise extracellular wiring while other cells within the multicellular circuit do not comprise extracellular wiring.
  • a multicellular circuit can involve wiring aspects and wireless aspects.
  • the output molecule produced by one or more cells within the multicellular molecular circuit can also be the final output molecule of the circuit.
  • the final output molecule of the circuit is detectable.
  • the final output molecule is a fluorescent molecule.
  • a fluorescent output signal is detected by FACS (FIG. 15).
  • FIG. 15 provides a non-limiting graphical demonstration of a reporter readout by FACS.
  • FIG. 15A and FIG. 15B yeast strains carrying the reporter GALl .GVP were grown with glucose (repressed conditions) or galactose (activated conditions).
  • FIG. 15C and FIG. 15D a 73 ⁇ 4t-ON: :GFP reporter was activated by adding doxicycline. GFP fluorescence in single cells is assessed quantitatively by FACS.
  • an input or output molecule is detectable if it is approximately 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10-fold higher than a control level.
  • a system can include at least two cells wherein the first cell is responsive to an external signal and the second cell is responsive to a signal (wire) produced by the first cell and optionally an external signal.
  • a system comprises three cells, wherein the first cell is responsive to an external signal and the second and third cells are responsive to one or more wiring molecules within the system and optionally one or more external signals.
  • a system comprises three cells, wherein the first and second cells are both responsive to external signals, which can be the same or different, and the third cell is responsive to one or more wiring molecules produced by the first and/or second cells and optionally one or more external signals.
  • the system comprises four or more cells wherein the first cell, and optionally one or more other cells, is responsive to an external signal and the remaining cells are responsive to wiring molecules within the system and optionally one or more external signals.
  • each cell within a system is responsive to a different input signal or combination of input signals.
  • more than one type of cell within a system is responsive to the same input signal or combination of input signals. It should be appreciated that responsiveness of a cell to a signal can in some embodiments mean that the signal causes activation of gene expression in the cell and in other embodiments mean that the signal causes inhibition of gene expression in the cell. In some embodiments, the same signal can cause activation of gene expression in some cells within the system and inhibition of gene expression in other cells within the system.
  • Computation such as Boolean computation
  • Boolean computation can be systematically implemented using the multicellular molecular circuits formed by multiple interconnected cells described herein. Each cell responds to one or more different input stimuli activating or inhibiting the production of a given output which can itself act as the input for another cell.
  • the methods described herein allow that the final output molecule of the circuit can be generated in more than one type of cell in the circuit, referred to herein as redundant distribution.
  • redundant distribution One of the advantages of redundant distribution of the desired output is the reduction of wiring constraints.
  • the distribution of logic functions in different cells allows for the reuse of the same molecular elements for performing different tasks.
  • the methods described herein allow the use and reuse of cells that comprise a particular logic function to implement different and complex functions such as Boolean functions.
  • computations are distributed among different cells.
  • a logic function can be a logic gate.
  • a "logic function” or a “logic gate” refers to a fundamental building block of a circuit.
  • Cells associated with the invention express genetic circuits.
  • a genetic circuit refers to a collection of recombinant genetic components that responds to one or more input molecules and performs a specific function, such as the regulation of the expression of one or more genes.
  • a logic gate can have one or more inputs and one or more outputs.
  • each input and/or output is represented by one of two binary conditions: low (0) or high (1).
  • an input or output can be measured as the identity of a molecule, the intensity (e.g., level) of output, the duration of expression or increased expression, or any combination thereof.
  • logic gates include AND, OR, NOT (also called INVERTER), NAND, NOR, IDENTITY, XOR, XNOR, IMPLIES and N-IMPLIES.
  • INVERTER AND, OR, NOT (also called INVERTER), NAND, NOR, IDENTITY, XOR, XNOR, IMPLIES and N-IMPLIES.
  • the operation of a logic gate can be described by a corresponding truth table.
  • "inverted logic” is exhibited.
  • inverted logic refers to an embodiment wherein a cell responds to one or more inputs by repressing output expression. Inverted logic can be used to increase the complexity and decrease the wiring within
  • An example of a logic gate is an AND logic gate with two inputs, wherein a positive output is produced only if both inputs are high.
  • a representative truth table for such a logic gate is as follows:
  • FIG. 2A A non-limiting example of an AND gate is shown in FIG. 2.
  • FIG. 2A demonstrates a cell which produces an output molecule only in the presence of two input molecules.
  • FIG. 2D presents a truth table corresponding to an AND gate.
  • FIG. 4A provides an example of a multicellular system wherein the cells are engineered to implement an AND logic gate. As is demonstrated in the truth table, only when the multicellular system is provided with two inputs, in this embodiment NaCl and 17 ⁇ - ⁇ 2 , does the system produce a positive output, which in this embodiment is GFP fluorescence.
  • the implementation of a logic gate such as an AND gate is achieved using a multicellular molecular circuit.
  • the multicellular molecular circuit comprises two cells with a combined function that corresponds to an AND logic gate.
  • Cell #1 responds to one of the input signals and produces an output molecule.
  • Cell #2 responds to the output molecule produced by Cell #1 and another input signal.
  • a positive final output signal from this multicellular molecular circuit is only produced in the presence of both Cell #1 and Cell #2 and in the presence of both input signals.
  • FIG. 4C provides a non-limiting example of a multicellular molecular circuit comprising three cells with a combined function that corresponds to an OR gate.
  • the presence of either input, NaCl or GAL leads to a positive output of GFP fluorescence.
  • NOT logic gate also called an INVERTER logic gate
  • the output is an inverted version of the input.
  • FIG. 2E A truth table corresponding to a NOT gate implemented in a multicellular molecular circuit is shown in FIG. 2E, wherein the response in an engineered cell is the inverse of the input.
  • a NAND gate is the equivalent of a "NOT-AND” gate, meaning an AND gate followed by a NOT gate. In an NAND gate, the output is high if any of the inputs are low.
  • a representative truth table for a 2-input NAND gate is as follows:
  • FIG. 4D A non-limiting example of a multicellular molecular circuit comprising an NAND gate is depicted in FIG. 4D. As indicated in the truth table, an output signal of GFP fluorescence is produced when one or both input signals, which in this embodiment are DOX and GLU, are low.
  • NOR gate is the equivalent of a "NOT-OR” gate, meaning an OR gate followed by a NOT gate. In this logic gate, output signal production will be low if any of the inputs are high.
  • a representative truth table for a 2 input NOR gate is as follows:
  • FIG. 4B A non-limiting example of a multicellular molecular circuit comprising an NOR gate is depicted in FIG. 4B.
  • an output signal which in this embodiment is GFP fluorescence, is produced only when levels of input signals, which in this embodiment are Dox and 6a, are low.
  • IDENTITY logic gate In an IDENTITY logic gate, a positive output is produced if an input signal is present.
  • a representative truth table for such a logic gate is as follows:
  • FIG. 2E A truth table corresponding to a cellular IDENTITY gate is demonstrated in FIG. 2E.
  • An XOR gate is also called an exclusive-OR gate.
  • the difference between an XOR gate and an OR gate is that in an XOR gate with two inputs, when both inputs are high or " 1", the output is low or "0.”
  • a representative truth table for such a logic gate is as follows:
  • An XNOR gate is the inverse of an XOR gate.
  • An XNOR logic gate is implemented with two inputs, a positive output is produced if both of the inputs are the same, meaning both inputs are "1" or "0.” If one input is high and the other is low, then no output is produced.
  • a representative truth table for such a logic gate is as follows:
  • FIG. 8C A non-limiting example of a multicellular molecular circuit exhibiting an IMPLES function is shown in FIG. 8C.
  • the multicellular circuit comprises three cells with input molecules being GLU and NaCl. A final output molecule is produced except in instances where GLU is high and NaCl is low.
  • N-IMPLIES logic gate In a non-limiting example of an N-IMPLIES logic gate, the output is high if input 2 is high but input 1 is low.
  • a representative truth table for such a logic gate is as follows:
  • FIG. 8B A non-limiting example of a multicellular molecular circuit exhibiting an N- IMPLES function is shown in FIG. 8B.
  • the multicellular circuit comprises two cells with input molecules being DOX and 17 ⁇ - ⁇ 2 .
  • a final output molecule will not be produced unless the DOX input is low and the 17 ⁇ - ⁇ 2 input is high.
  • Multicellular molecular circuits that comprise logic gates described above are non-limiting.
  • Multicellular molecular circuits described herein can comprise varying numbers of cells, and can utilize a range of input and output molecules or other signals.
  • compositions comprising two or more different types of recombinant cells wherein each type of recombinant cell comprises a logic gate.
  • the two or more different types of recombinant cells each comprise a different logic gate, while in other embodiments, more than one of the different types of recombinant cells comprise the same logic gate.
  • the two or more different types of recombinant cells each respond to a different input signal or combination of input signals, while in other embodiments, more than one of the different types of recombinant cells responds to the same input signal or combination of input signals.
  • at least two of the different types of recombinant cells produce the same output.
  • At least two of the different types of recombinant cells produce the same output signal that modulates the expression of one or more genes in one or more additional recombinant cells. At least two of the different types of recombinant cells can produce the same output signal that is detectable and used to determine the overall output of a computation system. In some embodiments, two or more subgroups of the different types of recombinant cells are spatially separated from each other, as discussed further below.
  • the multicellular distributed computation circuits are not spatially separated and do not involve wiring.
  • each cell responds to two different sets of inputs: those that induce an activity such as protein expression; and those that prevent the same activity, such as repressing protein expression by expressing a repressor.
  • the internal architecture of each cell is constant. When all inducing inputs are present and all repressor inputs are absent (the number varies depending on the implementation), a positive output will be produced, as described in FIG. 48.
  • a circuit comprises a "logic block” or a “logic module.”
  • logic block and “logic module” are used interchangeably to refer to a subpart of a logic circuit in which entries and exits are defined and in which the subpart implements a different logic function than that which is implemented by the whole circuit.
  • compositions and methods described herein relate to methods involving providing one or more input signals to a composition comprising two or more different types of recombinant cells, as described herein, and measuring the presence or absence of an output signal.
  • any of the outputs can be quantified or measured and put into a general purpose computer.
  • the method is a method for conducting biological computation.
  • the method involves converting an output signal into a digital signal.
  • Recombinant cells described herein can also be combined to generate more complex functions and circuits. For example, FIG.
  • FIG. 5 demonstrates the design and in vivo implementation of a multiplexer (MUX2al) and 1-bit adder with carry using recombinant cells described herein.
  • FIG. 5B presents a schematic of in vivo implementation of the MUX2tol circuit via distributed computation in recombinant cells.
  • FIG. 5C demonstrates in vivo implementation of the 1-bit adder with carry.
  • An advantage of the methods and compositions described herein, using distributed computation, is the ability to reprogram circuits, as demonstrated for example in FIG. 9.
  • FIG. 9A presents AND gate reprogramming including a schematic representation of cells used in the AND circuit, and a corresponding truth table with a third input to reprogram the circuit.
  • FIG. 9B presents OR gate reprogramming including a schematic representation of cells and a corresponding truth table.
  • aspects of the invention relate to the use of multicellular circuits as environmental sensors. Such circuits can respond to a variety of environmental stimuli such as biochemical, electromagnetic, radiation, light, heat and/or electrical stimuli.
  • multicellular circuits can be used in "artificial sensing.” As used herein, “artificial sensing” refers to reproducing human senses.
  • multicellular circuits can be used as an artificial nose to detect odors.
  • Artificial noses have widespread applications, including in research and development, such as: formulation or reformulation of products, benchmarking with competitive products, shelf life and stability studies, selection of raw materials, packaging interaction effects, simplification of consumer preference test; in quality control applications such as: conformity of raw materials, intermediate and final products, batch to batch consistency, detection of contamination, spoilage or adulteration, origin or vendor selection and monitoring of storage conditions; and in process and production for such uses as: managing raw material variability, comparison with a reference product, measurement and comparison of the effects of manufacturing process on products, following-up cleaning in place process efficiency, scale-up monitoring and cleaning in place monitoring.
  • multicellular circuits can be used as artificial tongues.
  • Artificial tongues have a variety of applications including: analyzing the ageing of flavors in beverages, quantifying a taste such as bitterness or "spiciness" of a compound, evaluating taste masking qualities of formulations, and analyzing stability of formulations.
  • multicellular circuits described herein include detection of microbes such as pathogens, including, for example, bacterial, viral and fungal pathogens.
  • a multicellular circuit may be used in a medical environment such as a hospital to detect contagious factors, or in an environment where food is processed or prepared as a means of quality control.
  • a multicellular circuit may be used to detect drugs or hazardous substances.
  • An example of a sensory network is presented in Example 5, which demonstrates regulation of glucose levels.
  • kits comprising recombinant cells.
  • a kit comprises one or more pairs of cells wherein each pair of cells responds to one or more different input signals.
  • one cell comprises an IDENTITY function and the other cell comprises an INVERTER (NOT) function.
  • NOT INVERTER
  • each different pair of cells produces the same output molecule in response to different input molecules.
  • one or more pairs of cells produce different output molecules.
  • the kit can comprise a reporter cell which responds to the output molecule produced by the pairs of recombinant cells and produces a final output signal.
  • the final output signal produced by the reporter cell can be detectable.
  • the final output signal is fluorescent.
  • a fluorescent signal can be detected by standard methods such as FACS or microscopy, familiar to one of skill in the art.
  • FIG. 14 depicts a library of engineered cells and a universal reporter strain. Identity and inverter logic functions are represented as (Id) and (NOT) cells. Inputs are indicated in each pair of cells. All of the cells have the same output (a-factor). The universal reporter is inhibited by a- factor.
  • Cells associated with the invention can be engineered to respond to at least one signal, at least two signals, at least three signals, at least four signals, at least five signals, or more than five signals.
  • at least one of the signals is a cell-generated signal or wire within the multicellular system.
  • at least one of the signals is an external signal, not produced by one of the cells within the multicellular system.
  • one or more promoters of one or more genes is engineered to be responsive to one or more of the signals described herein, wherein the promoter controls the expression of one or more output molecules (e.g., signal molecules and/or detectable output molecules).
  • FIGs 11, 12, 13, 16 and 45 Further aspects of the invention relate to spatial separation of cells or groups of cells within multicellular molecular circuits.
  • Non-limiting examples of spatial separation of cells or groups of cells are demonstrated in FIGs 11, 12, 13, 16 and 45. It should be appreciated that any means of spatially separating cells or groups of cells is compatible with aspects of the invention.
  • the cells are spatially separated through the use of a microfluidic device.
  • within a microfluidic device cells of different types are separated by membranes that are permeable to diffusible molecules such as wires transmitted between cells within the multicellular systems described herein.
  • Microfluidic devices compatible with methods and compositions described herein can be of any suitable make or manufacture, as would be understood by one of ordinary skill in the art.
  • cells are segregated into different chambers of a microfluidic device but the cells can communicate between the different chambers of the microfluidic device.
  • the microfluidic device is configured to provide nutrients and/or remove waste in order to maintain the viability of the one or more cell types. It should be appreciated that for each cell type a sufficient number of identical cells (e.g., a clone of cells) is provided to perform the functions described herein (e.g., responsiveness to an external input or system signal, production of a system signal and/or detectable output).
  • FIG. 1 IB depicts a schematic representation of a microfluidic plate, as a non- limiting example of spatial separation of cells within a multicellular circuit.
  • cell mixtures are loaded into wells of a microfluidic device.
  • Application of pressure in the cell wells introduces the cells into the microfluidic chamber where they are retained.
  • One or more inputs are introduced into input wells within the microfluidic device. Cells that are exposed to one of more inputs in the microfluidic chamber can be imaged, leading to output detection.
  • FIG. 45 depicts implementation of spatial separation in a microfluidic device.
  • FIG. 45A provides a schematic demonstrating the introduction of mixed input molecules into chambers containing different types of cells and the production of an output molecule.
  • FIG. 45B demonstrates that spatial separation of cells within the multicellular circuit allows for simplification of circuit implementation.
  • the cell can be a eukaryotic or prokaryotic cell.
  • the cell is a fungal cell such as a yeast cell, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains.
  • the yeast strain is a S.
  • fungi include Aspergillus spp., Pennicilium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
  • the cell is a bacterial cell, such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp.,
  • the cell is an algal cell, a plant cell, an insect cell or a mammalian cell.
  • the mammalian cell is a human cell.
  • multicellular systems described herein contain cells that originate from more than one different type of organism.
  • the multicellular molecular system can comprise one or more yeast cells and one or more cells from an organism other than yeast, such as one or more mammalian cells.
  • aspects of the invention relate to recombinant expression of one or more genes encoding components of logic functions and molecular circuits. It should be appreciated that some cells compatible with the invention may express an endogenous copy of one or more of the genes associated with the invention as well as a recombinant copy. In some embodiments, if a cell has an endogenous copy of one or more of the genes associated with the invention, then the methods will not necessarily require adding a recombinant copy of the gene(s) that are endogenously expressed.
  • cell(s) that recombinantly express one or more genes comprising logic functions associated with the invention and the use of such cells in biological computation are provided.
  • the genes associated with the invention can be obtained from a variety of sources. For example, genotypes of cells and strains that were used in the Examples section are presented in Tables 1 and 2. However, it should be appreciated that any genes that can produce signals and/or respond to signals can be engineered to be expressed at suitable levels for multicellular networks as described herein.
  • cells may be engineered to express one or more suitable receptors, transcription factors, transcriptional activators, transcriptional repressors, translation factors, translational activators, translational inhibitors, factors involved in mRNA or protein degradation, factors involved in RNA interference (RNAi), enzymes, ligands, etc., or any combination thereof.
  • suitable receptors such as STE2 and CaSTE2
  • MAPKinases such as HOG1 and FUS3
  • transcription factors such as GAL4 or DOS regulated transcription factors
  • Tta/rTta transcription factors
  • proteases for signal peptide processing such as BAR1 and KEX2
  • specific genes for pathway hyper- sensitization such as SST2 and FPS1.
  • any of the nucleic acids and/or polypeptides described herein can be codon-optimized and expressed recombinantly in a codon-optimized form.
  • homologous genes for any of the genes described herein could be obtained from other species and could be identified by homology searches, for example through a protein BLAST search, available at the National Center for Biotechnology Information (NCBI) internet site (ncbi.nlm.nih.gov).
  • Genes associated with the invention can be PCR amplified from DNA from any source of DNA which contains the given gene.
  • genes associated with the invention are synthetic. Any means of obtaining a gene associated with the invention are compatible with the instant invention. Aspects of the invention encompass any cell that recombinantly expresses one or more components of a logic gate and/or genetic circuit as described herein.
  • a "vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA, although RNA vectors are also available.
  • Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis.
  • replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • a variety of transcription control sequences can be used to direct its expression.
  • the promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene.
  • the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene.
  • conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • a nucleic acid molecule that comprises a gene associated with the invention can be introduced into a cell or cells using methods and techniques that are standard in the art. In some embodiments, it may be advantageous to use a cell that has been optimized for expression of one or more genes.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule can also be accomplished by integrating the nucleic acid molecule into the genome.
  • Codon usages for a variety of organisms can be accessed in the Codon Usage Database (http://www.kazusa.or.jp/codon/).
  • Protein engineering can also be used to optimize expression or activity of a protein.
  • a protein engineering approach could include determining the three dimensional (3D) structure of a protein such as an enzyme or constructing a 3D homology model for the protein based on the structure of a related protein. Based on 3D models, mutations in a protein can be constructed and incorporated into a cell or organism, which could then be screened for an increased or decreased production of a protein.
  • a nucleic acid, polypeptide or fragment thereof described herein can be synthetic.
  • synthetic means artificially prepared.
  • a synthetic nucleic acid or polypeptide is a nucleic acid or polypeptide that is synthesized and is not a naturally produced nucleic acid or polypeptide molecule (e.g., not produced in an animal or organism). It will be understood that the sequence of a natural nucleic acid or polypeptide (e.g., an endogenous nucleic acid or polypeptide) may be identical to the sequence of a synthetic nucleic acid or polypeptide, but the latter will have been prepared using at least one synthetic step.
  • Example 1 Distributed Biological Computation with Multicellular Engineered Networks
  • FIG. 2A-C A library of engineered cell types with restricted connections among them was generated in which each cell type responds to one or two different inputs (FIG. 2A-C).
  • each cell acts as a minimal logic block.
  • FIG. 2A illustrates a cell which receives a signal from another cell (IN) and a signal from an external source (E).
  • FIG. 2B illustrates a cell that just receives a signal from an external source. Both cells produce a diffusive output molecule.
  • FIG. 2C presents a schematic representation of cell behavior, where each cell cy responds to two different inputs namely an external input Xj and an internal wire ⁇ 3 ⁇ 4. ⁇ .
  • the response of k-th cell type o(k;i j) can be the production of a new internal ⁇ 3 ⁇ 4 wire or the final output.
  • the basic two-input and one-output engineered functions include the AND and the inverted IMPLIES, named N-IMPLIES (FIG. 2D). These two functions allow any other Boolean function to be implemented. Moreover, some cells can be simplified by implementing one-input, one-output functions (FIG. 2E).
  • the output of each cell type is a diffusible molecule that can either transmit the signal to a neighboring cell (acting as a cell-to-cell wire) or can act as the final system's output. In some embodiments, only unidirectional cell-to-cell-cell communication is used.
  • FIG. 3 A general-purpose multicellular network is shown in FIG. 3. The computation is determined by: (1) the number of cells involved, represented by "C,” (2) the specific function implemented by each engineered cell (i.e. AND or N-FMPLIES) and (3) the location of cells within the network (see Example 2 and FIG. 6). As described above, using a minimal set of cells, the network is built with the premise that, in principle, cells are connected in a single direction, i.e., cell (3 ⁇ 4. / connects with cell c y - but not vice versa (FIG. 3).
  • FIG. 3 presents a schematic representation of the general architecture of multicellular circuits with a fixed wiring pattern.
  • Each set p t contains N different cells at the most, i.e c i2 , ... c iN ) .
  • N is the number of inputs of the Boolean function.
  • Cells in the same set > are connected according to a fixed wiring pattern shown in FIG. 2C. Every set >, is able to produce the final system' s output O independently, i.e. there are no connections between cells from different sets.
  • FIG. 6 demonstrates a method for designing distributed biological computation.
  • FIG. 6A presents a truth table defining the behavior of a multiplexer circuit.
  • FIG. 6B presents a full Boolean function that expresses the relationship between the logic inputs xl, x2, x3 and the logic output O according to the truth table.
  • FIG. 6C presents a simplified version of the Boolean function / describing the same truth table.
  • the analytical expression of the Boolean function determines that the circuit can be implemented using two different sets of engineered cells pi and p2.
  • FIG. 6D demonstrates implementation of the circuit with direct mapping between the terms of the Boolean function and the engineered cells.
  • FIG. 6E demonstrates a mixed implementation, where the set pi is a direct mapping of the terms from the Boolean function whereas in p2 all the terms have been condensed in a single cell.
  • FIG. 6F demonstrates circuit implementation where both sets pi and p2 are implemented by a single cell in each case.
  • This cell to cell communication scheme is different from previous approaches to biological computation.
  • different types of engineered cells can produce the final output of the computation and thus, computation is distributed and results from the combination of different strings of cells (FIG. 3).
  • each cell activates the response of the next one via cell-cell communication.
  • This cascade of activations can be interpreted as a carrier signal, which is modulated by external inputs, e.g., each external input can trigger a decision, which can be, for example, that the carrier signal flows towards the next cell in the string or the carrier signal is blocked.
  • Reprogramming can be easily achieved by the addition or modification of a specific cell type into the circuit. This is rather different from previously described single-cell approaches, where even a moderately complex circuit needs to be implemented in a specific way and is unlikely re-usable for another task.
  • a feature of the multicellular approach described herein is that once a library of engineered cells has been constructed, it is possible to easily combine them in multiple ways to implement different circuits.
  • FIG. 7 presents graphs demonstrating the number of possible Boolean functions versus the number of different cells required for their implementation. Each graph represents the number of (non-null) functions that can be implemented with a defined number of engineered cells that receive 2-inputs (FIG. 7A) and 3- inputs (FIG. 7B).
  • the output of the cells was monitored as the expression of a reporter construct under the control of the pheromone responsive promoter FUS1 (e.g. GFP).
  • FUS1 pheromone responsive promoter
  • FIG. 23 The relevant genotype and the graphical notation that indicates the logic function performed by each cell of the library is depicted in FIG. 23.
  • the ability of cells to respond to external stimuli was monitored by fluorescence in single cell by FACS and referred to a reference of the maximal number of cells able to produce output signal (an example of a NOT cell is presented in FIG. 20A).
  • Each cell of the library was characterized for its ability to respond to the corresponding stimuli (transfer function) to one or two external inputs.
  • An example of this quantification for cells with the basic logic functions is presented in FIG. 20B (transfer function for the engineered cells is included as FIG. 24). It should be appreciated that the principles exemplified in the Examples are non-limiting and can be implemented with different cell types, input and output signals, wiring molecules, or any combination thereof as described generally herein.
  • FIG. 4A presents an example of a cellular AND gate and corresponding truth table.
  • the genotypes of the cell strains and plasmids are presented in the Examples section. Cells were grown in YPD to mid-exponential phase and washed twice with YPD. The cells were mixed proportionally, and inputs (NaCl and 17P-estradiol) were added at the same time. Cells were then incubated for 4h at 30 °C and analyzed by flow cytometry as described in the Examples section. Data is expressed as the percentage of GFP positive cells (considering 100% an equally treated sample with 2 ⁇ g/ml of alpha factor).
  • FIG. 4B presents an example of a cellular NOR gate and corresponding truth table. Cells were treated as in FIG. 4A using as inputs 10 ⁇ g/ml doxycycline and 5 ⁇ 6a.
  • FIG. 4C presents an example of an OR gate and corresponding truth table. Cells were treated as in FIG. 4A using as inputs 0.4 M NaCl and 2 % galactose.
  • FIG. 4D presents an example of a cellular NAND gate and corresponding truth table. Cells were treated as in FIG. 4A using as inputs 10 ⁇ g/ml doxycycline and 2 % glucose.
  • this example illustrates a non-limiting design of a basic circuit with an AND logic (FIG. 4A).
  • This circuit included two basic engineered cells that respond to two different stimuli (NaCl and estradiol) and use pheromone (alpha factor) as a wiring molecule.
  • the presence of NaCl stimulates Cell #1 to produce alpha factor that is secreted into the media (IDENTITY) that is received by Cell #2.
  • Cell #2 has the ability to sense another external input (estradiol) and it is competent, via the production and activation of the Fus3 MAPK, to produce the final output (induction of a fluorescent reporter molecule, FUS1..GFT*).
  • FUS1..GFT* fluorescent reporter molecule
  • the resulting AND circuit has a well defined separation between 1 and 0 logic with respect to the typical resolution of electronic devices.
  • the family of TTL devices 17 works in a range from 0 to 5 volts. A voltage below 0.5 volts is considered 0 logic, whereas voltages above 3 volts (up to 5 volts) are 1 logic.
  • 0 logic is less than 10% of the maximal voltage.
  • FIG. 4 shows, in these biological devices, the resolution of the 0 logic is less than 10% of the maximal value, indicating that these circuits are comparable with electronics in terms of resolution.
  • using populations of cells instead of a single cell to implement the circuit allows for a drastic reduction of the noise as well as the stochastic fluctuations, providing a stable, well-defined output.
  • a circuit was then implemented with an NOR logic.
  • the engineered cells to implement this circuit are different from those previously used since they require a NOT function instead of an IDENTITY function, and an N-IMPLIES function instead of an AND function.
  • a two cell circuit was implemented in which each cell responded to a particular stimulus (Doxicycline and 6a, an inhibitor of the fus3as kinase) and pheromone was used as a wiring molecule.
  • NOR gate only in the absence of both stimuli was there a positive final outcome (FIG. 4B).
  • AND and NOR circuits To implement these two circuits, i.e. AND and NOR circuits, a full set of engineered cells were developed that can be used to construct virtually any Boolean function.
  • the NAND gate was constructed using cells already implemented for the NOR and OR circuits. Other circuits (such as N-FMPLIES and IMPLIES) can also be constructed through reuse (FIG. 8). Thus, a major advantage of distributed computation is the re-utilization of simple engineered cells as basic modules. Of note, it is possible to design and implement a dedicated N-IMPLIES logic circuit in a single cell (FIG. 8A) or alternatively, by combining cells with different logics used in previous circuits (FIG. 8B).
  • FIG. 8 demonstrates implementation of N-IMPLIES and FMPLIES circuits.
  • FIG. 8A presents a schematic representation of a cell with an N-IMPLIES logic and the corresponding truth table. The cells depicted in this schematic respond to doxycycline and estradiol inputs. Data are expressed as the percentage of GFP positive cells (considering 100% an equally treated sample with synthetic alpha factor).
  • FIG. 8B presents a schematic representation of N-IMPLIES logic implemented in two cells with different logic.
  • FIG. 8C presents a schematic demonstration of cells described in FIG. 4C used to implement an FMPLIES logic circuit. Inputs were Glucose and NaCl.
  • FIG. 25A To further characterise the basic computational properties of engineered systems described herein, the AND circuit presented in FIG. 25A was analysed as a reference. Cellular response to dynamic inputs, stability of the network and the ability of the AND circuit to respond for long periods of times were investigated. It was found that once the circuit is turned on, it can maintain maximal signal for periods beyond 9 hours (four generations) in the presence of stimuli (FIG. 26A). Furthermore, once the network of cells has been established, it responds equally well for long periods of time (at least four generations) while the culture is maintained to a log phase (FIG. 26B). The responsiveness of the network to dynamic changes was also addressed.
  • a microfluidic device was set up containing the cells of the AND circuit and the cells were exposed to changes in the input signals over time. Only in the presence of the two inputs, was GFP production induced (FIG. 26C). After inactivation and photobleaching to eliminate previously produced GFP, the circuit was able to respond again to stimuli (FIG. 26C).
  • An additional advantage of this type of biological circuit is that the systems have been engineered such that they can be selectively switched off or reprogrammed.
  • This capability permits for modification of the response of part of the cells involved in the circuit, allowing for in vivo reprogramming. For instance, the inhibition of the intracellular signal transduction in Cell #2 of the AND gate, blocks the positive outcome of the circuit (FIG. 9A).
  • reprogramming is performed in more complex circuits containing a larger number of cells, different computations can be achieved. For instance, when a reprogramming molecule (such as glucose) is added to the OR gate shown in FIG. 9, this changed its computation to an IDENTITY function for NaCl (input 2).
  • FIG. 9 demonstrates circuit reprogramming and associated data.
  • FIG. 9A presents AND gate reprogramming including a schematic representation of cells used in the AND circuit, and a corresponding truth table with a third input to reprogram the circuit.
  • x represents any possible value, i.e. 0 or 1.
  • Indicated cells were treated as in FIG. 4A and inhibitor (6a) was added to reprogram the indicated samples.
  • Data are expressed as the percentage of GFP positive cells (considering 100% an equally treated sample with 2 ⁇ g/ml of synthetic alpha factor). Results are presented as the mean ⁇ SD of three independent experiments.
  • FIG. 9B presents OR gate reprogramming including a schematic representation of cells and a corresponding truth table. 2% Glucose was added to reprogram the indicated samples.
  • Cell #7 responds to an alternative pheromone from C. albicans, which acts as independent signaling molecule.
  • Cell #3 and Cell #2 respond to doxycicline and estradiol respectively (and were described above), whereas Cell #7 responds to doxycicline and C. albicans pheromone.
  • the final output (GFP) is generated by Cell #2 and Cell #7.
  • the complexity of the circuit involved a differential output to eight different input combinations, the in vivo results clearly showed that the computation of the three inputs yielded the expected output response (FIG. 5B).
  • FIG. 5 presents the design and in vivo implementation of a multiplexer (MUX2al) and 1-bit adder with carry.
  • FIG. 5 A presents a schematic diagram of a putative transcription based single cell MUX2al .
  • FIG. 5B presents a schematic of in vivo implementation of the MUX2tol circuit via distributed computation in engineered cells. The genotypes of strains and plasmids used are described herein. A truth table corresponding to a MUX2tol circuit is also presented. Cells were treated as in FIG. 4A using doxycycline (selector) and the inputs 17P-estradiol and synthetic Candida albicans alpha factor.
  • FIG. 5C demonstrates in vivo implementation of the 1-bit adder with carry.
  • Four cells that respond to galactose and doxycycline with an XOR logic were combined with cells that respond to the same stimuli but with an AND logic (lower panel). The final outcome was measured as in FIG. 4A.
  • Results are presented as the mean ⁇ SD of three independent experiments. Black bars indicate the adder output whereas grey bars represent the carry bit.
  • MUX2tol circuit was implemented that contains four cells and uses two
  • output cells express different reporter proteins, a green reporter (adder) or a red reporter (carry) (FUS1::GFP or FUSl::mCherry respectively) which permits to monitor the outcome of the carry and adder in the same culture.
  • To implement the XOR gate four different engineered cells that responded to two inputs (doxycycline and galactose) were combined. As expected for an XOR gate, only in the presence of one of the stimuli was the outcome positive (FIG.
  • FIG. 5C shows that the first circuit was implemented with the combination of four different cells. Combination of four cells greatly increases the potential for computations (FIG. 7).
  • the XOR was then used in combination with an AND gate that responded to the same stimuli but the cells were maintained in separated compartments. This spatial separation can be bypassed if a different wiring molecule and output are implemented, to construct the 1-bit adder with carry.
  • FIG. 5C shows that the systems respond as an XOR gate (black columns) but the presence of the two stimuli induces the presence of the 1-bit carrier (grey columns). Therefore, taking advantage of the enormous combinatorial potential of simple constructs, reprogrammable, complex circuits can be implemented that can be seen as instances of a LEGO ® -like combinatorial system (FIG. 10).
  • FIG. 10 presents a schematic summary of eight examples of logic gates implemented by single or multicellular components described herein, represented as LEGO®-like structures.
  • the cellular components can be used in multiple ways in order to generate desired (non-trivial) Boolean functions. Adding an additional type of wire or another output molecule causes the number of constructs to exponentially increase. Aspects of the distributed computation approach described herein can be easily extended to other model organisms and be implemented through appropriate engineering of transcriptional, translational or post-translational control elements.
  • W303 (ade2-l his3-ll,15 leu2-3, 112 frpl-1 ura3-l canl) cells were genetically modified to produce alpha factor (wiring molecule) from an inducible promoter. Strains mfal and mfa2 were used to prevent endogenous production of alpha factor and produce pheromone under regulatable promoters. STE3 was also deleted to avoid mating between cells in the circuit. Output cells contained a BAR1 deletion to increase sensitivity to alpha factor. Some strains contain FUS3 and KSSI deletions to prevent signaling through the pheromone pathway unless inducible FUS3 was expressed. A fluorescent reporter protein was integrated in the FUS1 ORF locus for output detection. To use the C. albicans alpha factor as a wiring molecule, cells expressed a modified pheromone detectable by yeast expressing CaSTE2. A complete list of plasmids and strains is included in FIG. 23 and Tables 1 and 2.
  • YPD YPGal
  • SD synthetic dropout
  • yeast nitrogen base without amino acids, 0.5% ammonium sulfate, 2% glucose, Complete Supplement Mixture quadruple dropout (Qbiogene, Irvine, CA) (0.6 g/liter), and histidine, tryptophan, uracil, or leucine combined (40 mg/liter)
  • dox doxycycline
  • galactose induction/repression of promoters cells were grown overnight separately with or without the input.
  • Microscopy based microfluidic platform Microscopy based microfluidic platform.
  • Cells were loaded into Y04 plates (CellASIC, San Leandro, CA). Cell loading into a chamber was performed at 8 psi and constant flow was maintained at 5 psi during position selection. During experiments, flow was reduced to 1 psi to reduce washing out of the produced alpha factor. Images were automatically taken every 10 minutes using NIS elements Software (Nikon, Melville, NY) and a Nikon Eclipse Ti Microscope. Images were analyzed with Matlab.
  • digital circuits can be described by a more or less complex Boolean function / with N input bits and M output bits. This function describes how to determine the Boolean outputs from any set of possible inputs, namely:
  • ⁇ 0,1 ⁇ N and ⁇ 0, 1 ⁇ M indicate the set of all possible strings of N inputs and M outputs, respectively.
  • Every Boolean function, as defined here, can be implemented in multiple ways, by combining different sets of logic gates.
  • Such sets of logic gates are known as functionally complete sets.
  • One particular systematic implementation of an arbitrary logic circuit can be obtained by interconnecting a set of two particular binary gates (AND and OR) and the NOT gate 1 ' 2 . Therefore, it is feasible to systematically implant complex circuits by using the following functionally complete set:,
  • Boolean computation can be systematically implemented using circuits formed by multiple interconnected cells.
  • each cell responds to different input stimuli activating or inhibiting the production of a given output which can itself act as the input for another cell.
  • the methods described herein allow that the final output of the circuit can be generated in different cells of the circuit. Two main advantages arise from this circuit design; the redundant distribution of the desired output drastically reduces wiring constraints, and the distribution of the logic blocks in different cells allows the reuse of the same molecular elements for performing different tasks.
  • the methods described herein allow the use and reuse of engineered cells with a particular logical computation to implement different and complex Boolean functions.
  • ⁇ represents the OR operator and ⁇ the AND operator.
  • Q is the maximum number of terms that depends on the complexity of the function, but always the condition Q ⁇ 2 N_1 is satisfied 3 .
  • the expression of a Boolean function / can be reduced by the systematic application of standard rules of simplification, such as the so called Karnaugh maps or the Quine- McCluskey algorithm 2
  • N j i e s '' ij x j ) - 1 in the equation (1).
  • the number of cells is ⁇ , ⁇ depending of the specific function implemented.
  • every cell Cy produces an output a j responding both to an external input x, and to the output o3 ⁇ 4. 1 of the previous cell Cy.i according to the logic function ⁇ , a, H AND0* ij(x.)
  • the wiring pattern is unidirectionally fixed, namely the behavior of cell Cy is determined by the behavior of the cell Cy.i but Cy do not condition Cy.i.
  • the behavior of these cells can be described by the following truth tables:
  • N-IMPLIES inverted IMPLIES
  • the first cell Cu only responds to an external input xy because there is not a previous cell connected to it.
  • This particular case can be implemented in two different alternatives: i) with one input cell with the proper logic, i.e. IDENTITY or NOT respectively, or ii) with the addition of a cell 2 that responds to two external inputs X] and x 2 according to the behaviors described previously, i.e. AND or N- IMPLIES. Both possibilities have been implemented experimentally (see FIG. 5B).
  • FIG. 6 For illustrative proposes, one of the described method for designing distributed biological computation is shown in FIG. 6.
  • the circuit implemented is a multiplexer with 3 -inputs and 1 -output named MUX2tol .
  • FIG. 6B shows the Boolean function / implementing the truth table. This function can be simplified using standard methods 2 .
  • FIG. 6C shows the simplified function implementing the truth table.
  • FIG. 6D-F show several alternative options to implement the same logic function.
  • FIG. 6D shows a circuit obtained by direct mapping of the function shown in FIG. 6C.
  • each term of the function is implemented by a different cell.
  • FIG. 6D shows an intermediate example, where the terms of pi are implemented in different cells, but the terms of p 2 are condensed in a single cell.
  • FIG. 6E shows the opposite case, where the pi and p 2 have been condensed in a single cell each one. This example has been implemented experimentally (see FIG. 5). Despite that the example shown in FIG.
  • the number of different cells required is determined by two different constraints, namely, the number of inputs N and the intrinsic complexity of the Boolean function.
  • the set S(N,M) of N-input, -output Boolean expressions can be determined.
  • Considering Boolean functions with a single output ( l), it is possible to construct at the most
  • FIG. 7A-B show the total number of functions that can be implemented versus the number of cells required for their implementation. As shown by these statistical analyses, a significant increase in the number of functions that can be achieved can correspond to a small increase in the number of cells involved in the circuit.
  • each wire must be a different biochemical element, e.g., a different protein, in order to prevent undesired crosstalk.
  • FIG. 22 shows the number of different functions with 3 -inputs that can be implemented versus the number of required wires for their implementation.
  • FIG. 22A in the approach described herein, the wiring requirements are significantly lower than other standard approaches (FIG. 22B,C), ensuring an important increase in the scalability of the circuits' complexity. As an example, half of the possible functions are already available using just two wires and an additional wire allows for more than 80% of the possible spectrum.
  • Yeast W303 (ade2-l his3-ll,15 leu2-3, 112 trpl-1 ura3-l canl) cells were genetically modified. Schematic genotypic characteristics of each cell and plasmid used are summarized in Tables 1 and 2.
  • Cell#l is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous a-factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the MFal gene is under the control of the STL1 osmoresponsive promoter in the episomal plasmid pRS424STLl-MFal to express a- factor in the presence of NaCl.
  • YCplacl95-j «7zi/ plasmid encodes a constitutively open version of the Fpsl glycerol channel.
  • fpslAl mutation is used to increase sensitivity to high osmostress, and thus induce higher a-factor expression. This cell implements an IDENTITY function.
  • Cell#2 is a MATa cell that contains BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSSI are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • GFP was introduced in the FUSI gene locus under its promoter.
  • GALS:fus3as construct was integrated to regulate fus3as expression in galactose/glucose growing conditions.
  • GALS version of GAL promoter was used to prevent leakiness in glucose.
  • ADGEV construct encoding the hybrid transcription factor "GEV" (Gal4DBD-hER-VP16 fusion protein) under the control of the ADH1 promoter was also integrated to regulate GAL genes with 17P-estradiol. This cell implements an AND function with 17P-estradiol and an N-IMPLIES function with glucose as input in galactose based circuits.
  • Cell#3 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the MFal gene is expressed under the control of 2 TetOperators in the centromeric plasmid pCM183- Fa/ that also expresses the Tet Transactivator. This allows cells to repress ⁇ -factor expression in the presence of doxycycline. This cell implements a NOT function.
  • Cell#4 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSSI are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • GFP was introduced in the FUS1 gene locus under its promoter.
  • fus3as construct with its own promoter was integrated to regulate fus3as activity with 6a inhibitor. This cell implements an N-IMPLIES function.
  • Cell#5 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the MFal gene is expressed under the control of the GAL1 promoter in the episomal plasmid pBEVY-GU- Fa/ to express a-factor in galactose.
  • This cell implements an IDENTITY function upon galactose addition or a NOT function in glucose in galactose based circuits.
  • Cell#6 (reporter cell) is a MATa cell that contains a BAR1 deletion to increase ⁇ -factor sensitivity. GFP was introduced in the FUS1 gene locus under its promoter. This cell implements an IDENTITY function.
  • Cell#7 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity. FUS3 and KSSI are deleted to prevent activation of the mating pathway unless fus3as is expressed. GFP was introduced in the FUS1 gene locus under its promoter.
  • the fus3as gene under the control of 7 TetOperators in the episomal plasmid pRS413Tet07- 3 ⁇ 453a5 that also expresses the reverse Tet Transactivator was introduced to regulate fus3as expression in doxycycline.
  • STE2 is deleted to prevent S. cerevisiae - factor signaling.
  • CaSTE2 was expressed from the pAJlCaSTE2 plasmid to make cells competent for C. albicans ⁇ -factor signaling. This cell implements an AND function with doxycycline but with C. albicans ⁇ -factor as a wire.
  • Cell#8 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the MFal gene is expressed under the control of the glucose responsive promoter HXT1 in the episomal plasmid YEpHXTl- Fa/. This cell implements an IDENTITY function.
  • Cell#9 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSSI are deleted to prevent constitutive signaling ability.
  • GFP was introduced in the FUS1 gene locus under its promoter.
  • the fus3as gene under the control of seven TetOperators in the integrative plasmid Ylp IetOl -fus3as that also expresses the reverse Tet Transactivator, was introduced to regulate fus3as expression in doxycycline. This cell implements an AND function.
  • Cell#10 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the MFal gene is expressed under the control of 2 TetOperators in the centromeric plasmid YCpTet02- Fa7 that also expresses the reverse Tet Transactivator was introduced to regulate ⁇ -factor expression in doxycycline. This cell implements an IDENTITY function.
  • Cell#ll is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSSI are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • GFP was introduced in the FUS1 gene locus under its promoter.
  • the fus3as gene under the control of 7 TetOperators in the integrative plasmid YIpTetOff7 -fusSas that also expresses the Tet Transactivator was introduced to repress fus3as expression in doxycycline. This cell implements an N-IMPLIES function.
  • Cell#12 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSS1 are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • GFP was introduced in the FUSI gene locus under its promoter.
  • a GALS:. :fus3as construct was integrated and an ADGEV construct encoding the hybrid transcription factor "GEV" (Gal4DBD-hER-VP16 fusion protein) under the control of the ADH1 promoter was also integrated to regulate GAL genes with 17 ⁇ - estradiol.
  • GAL4 was deleted to prevent activation of GAL genes in galactose. This cell implements an AND function.
  • Cell#13 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 was deleted to prevent mating with MATa cells within the circuit.
  • the CaMFal gene was expressed under the control of 7 TetOperators in the episomal plasmid YEpTet0ff7- CaMFal that also express the Tet Transactivator.
  • the CaMFal gene contains the S. cerevisiae MFal signal peptide for secretion and proteolysis followed by just one copy of C albicans MFal peptide sequence. This allows cells to repress C albicans ⁇ -factor expression in presence of doxycycline. This cell implements a NOT function.
  • Cell#14 is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression.
  • STE3 is deleted to prevent mating with MATa cells within the circuit.
  • the CaMFal gene under the control of 7 TetOperators in the centromeric plasmid YCpTet07- CaMFal that also expresses the Tet Transactivator, was introduced to regulate ⁇ -factor expression in doxycycline.
  • the CaMFal gene contains the S. cerevisiae MFal signal peptide for secretion and proteolysis followed by just one copy of C albicans MFal peptide sequence. This cell implements an IDENTITY function.
  • Cell#15 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSS1 are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • GFP was introduced in the FUSI gene locus under its promoter.
  • a GALS::fus3as construct was integrated to regulate fus3as expression in galactose/glucose growing conditions.
  • the GALS version of the GAL promoter was used to prevent leakiness in glucose.
  • STE2 was deleted to prevent S. cerevisiae a-factor signaling.
  • CaSTE2 was expressed in the YIpCaSTE2 plasmid to make the cell competent for C. albicans ⁇ -factor signaling. This cell implements an AND function.
  • Cell#16 is a MATa cell that contains a BAR1 deletion to increase a-factor sensitivity.
  • FUS3 and KSS1 are deleted to prevent activation of the mating pathway unless fus3as is expressed.
  • FUSl::mCherry was integrated for different output production.
  • the fus3as gene under the control of 7 TetOperators in the integrative plasmid Ylp let01-fus3as that also expresses the reverse Tet Transactivator, was introduced to regulate fus3as expression in doxycycline. This cell implements an AND function.
  • ADGEV denotes a construct encoding the hybrid transcription factor "GEV” (Gal4DBD-hER-VP16 fusion protein) under the control of the ADH1 promoter [1], Table 2. Plasmids Used in This Study
  • a clear separation between the logic state 0 and 1 can be critical. This separation can be done defining a single threshold that separates states (as CMOS electronic devices do) or using two different thresholds, the low and the high (TTL electronic devices). In the second case, there is a gap between both thresholds, which corresponds to an undefined state.
  • the logic sates are much more robust in terms of noise fluctuations because changing the state requires crossing the gap region.
  • a 6-fold increase from the low to the high state is enough for a proper definition of the thresholds with a significant gap region.
  • the family of TTL devices 8 works in a range from 0 to 5 volts. A voltage below 0.5 volts is considered 0 logic, whereas voltages above 3 volts (up to 5 volts) are 1 logic. Hence, in TTL devices 0 logic is less than 10% of the maximal voltage. As shown in FIGs. 4, 8, 9, 20, 21 and 25, in the biological devices described herein, the resolution of the 0 logic is less than 10% of the maximal value, indicating that these circuits are comparable with electronics in terms of resolution. However, this separation between logic states is necessary but not a sufficient condition to guarantee that multicellular circuits can be implemented connecting different cells acting as logic blocks.
  • Characterization of the library of engineered cells allows for analysis of the so-called Transfer Curve, i.e. the cellular response with respect to different input levels.
  • An adequate transfer curve can be characterized by several features 9"10 , namely i) a step-like shape, ii) linear or higher gain ranges in order to ensure that the signal will not be degraded from input to output for in a single cell, iii) adequate noise margins, without overlap between the high and the low state, and iv) each cell responds properly to the specific inputs and must ignore the rest of inputs of the circuit.
  • FIG. 24 shows the full set of transfer curves for each cell. All of these curves exhibit the proper shape to be logic blocks for a multicellular implementation. In each 1 -input 1 -output cell the gain has been calculated 10 . All the functions have a gain above 7.6, which guarantees the maintenance of the signal in all the circuits.
  • Cells produce a diffusible molecule (alpha-factor) that acts as a wire. To detect the secretion of these molecules, producer cells were mixed with a reporter cells expressing GFP upon alpha-factor. This procedure allows for characterization of the cellular behavior and also the wire efficiency.
  • the FACS data presented in FIGs. 27-34 displays the single cell measurements of output production upon stimuli.
  • FIGs. 35-42 show the individual cellular response of each cell in response to the different inputs they encounter within a circuit.
  • the experimental data clearly demonstrate that there are no undesired cross-talks and each cell responds only to the expected input.
  • circuits that contain two-wiring molecules e.g., alpha factor from S. cerevisiae and from C. albicans
  • Example 3 Implementation of microfluidic devices as a platform for spatially restricted computation
  • Spatial confinement of cells into connected compartments dramatically reduces circuit complexity and wiring requirements. This can be achieved by the use of spatially restricted devices such as microfluidics (El-Ali et al., 2006; Keenan and Folch, 2008). Spatial separation enables complex circuits to be easily constructed, expanding the potential for building cell-based computers.
  • FIG. 11 A A non-limiting example of a spatially distributed, multicellular implementation is provided in FIG. 11.
  • three different chambers contain (overall) only four different types of engineered cells, each one (indicated with different symbols) responding to a given input which leads to the production or not of a given output signal (a) that will connect the signal to a reporter cell.
  • Each chamber contains the same reporter cell, responding to the input signal molecule and producing an output molecule defining the chamber-level output. The presence or absence of this output defines the global circuit's response.
  • Input molecules can be organic or inorganic. Available technology can be used to provide microfabrication techniques for implementation of such devices and more complex devices including hundreds of channels and chambers.
  • FIG. 12 demonstrates examples of the implementation of two types of circuits in microfluidic devices.
  • a multiplexor, shown in FIG. 12 A in standard electronics involves four different logic gates and six wires.
  • such a circuit in microfluidics with spatial confinement involves only five types of engineered cells.
  • a representative corresponding truth table is shown in FIG. 12C.
  • the engineered cells include: an NaCl repressed cell, a Dox activated cell, a Gal repressed cell, a Dox repressed cell and an inverted reporter cell.
  • two chambers are used, each one containing two different types of the engineered cells plus the reporter cell.
  • Each engineered cell responds to a given input (such as NaCl, Doxycicline or Galactose) which leads to the production or not of a given output signal (such as a-factor).
  • a given input such as NaCl, Doxycicline or Galactose
  • Both chambers contain the same reporter cell that responds to the input signal molecule (such as a-factor) and produces an output molecule (GFP), defining the chamber-level output. The presence or absence of this output defines the global circuit's response.
  • one chamber contains (1) a cell responding to NaCl input (NaCl repressed cell), (2) a cell responding to Doxycicline input (Dox activated cell) and (3) the inverted reporter cell.
  • the second chamber has (1) a cell responding to Galactose carbon source (Gal repressed cell), (2) a cell responding to Doxycicline input (Dox repressed cell) and (3) the inverted reporter cell.
  • the Dox activated cell is a MATa cell that contains MFal and MFa2 deletions to avoid expression of endogenous ⁇ -factor expression and an STE3 deletion to prevent mating with MATa cells within the circuit.
  • the MFal gene under the control of 2 TetOperators in the centromeric plasmid YCpTet02- Fa7 that also expresses the reverse Tet Transactivator is introduced to regulate ⁇ -factor expression in doxycycline.
  • FIG. 12B depicts a comparator, which is a more complex circuit involving eighteen logic gates and sixteen wires.
  • FIG. 13 demonstrates the re-programmability of cellular circuits in microfluidic devices.
  • FIG. 13 A demonstrates microfluidic implementation of a function described by the corresponding truth table.
  • FIG. 13B shows the implementation of a different function and corresponding truth table. In this embodiment, re-programming the device only requires relocating some cells in the compartments and removing one of them out of the system, not re-engineering any of the cells.
  • General purpose microfluidic devices can be utilized to implement multiple channels for combinatorial mixing. Engineered cells are distributed into different compartments where they grow, interact under given sets of inputs and respond to them in predefined ways, allowing for computation. Available technology can be used to provide microfabrication techniques for implementation of such devices and more complex devices including hundreds of channels and chambers. Commercially available microfluidic devices are available, for example, from CellASIC, Hayward, CA. Microfluidic devices are also combined with advanced light microscopy as a platform for complex cellular computation.
  • Microfluidic devices can be used to generate efficient self-configurable cellular systems.
  • inputs can be externally applied and corresponding cells can produce two different kinds of response, namely the cell response is generated in presence of input (identity function) or in absence of input (inverse function). Combining these two types of cells in different chambers allows a variety of computations to be performed.
  • both types of cells can be present in the device simultaneously.
  • an external instruction specific stimuli
  • the system can be configured such that only one of two possible cells for each input would be operative. Thus, this would result in the appropriate configuration of cells to implement the expected function.
  • the external instruction signal
  • the system will change its configuration towards the new functionality required.
  • FIG. 16 provides a schematic representation of self-configurable systems.
  • FIG. 16A depicts a system for functions with one-input, one-output.
  • I 0 is the input and Co is the reconfiguration signal.
  • C 0 allows a choice between identity and inverted functions.
  • Co and Ci addition the equilibrium between both types of cells changes, blocking one of them. Once the new configuration is reached, the external signals can be removed and the system will remain configured.
  • Cells are grown in synthetic medium containing: 0.17% Yeast Nitrogen Base without amino acids, 0.5% ammonium sulfate, 2% of a carbon source (such as glucose, galactose or raffinose), 0.6g/l of Complete Supplement Mixture quadruple dropout, Histidine, Leucine, Tryptophan or Uracil (40mg/l). Distilled water can be autoclaved and stock solutions can be filter sterilized to avoid medium autofluorescence. Cells are grown separately at 30°C and maintained in exponential phase overnight. Cells are washed twice with fresh medium and mixed in the different chambers at equal proportions (final OD 0.2).
  • a carbon source such as glucose, galactose or raffinose
  • Distilled water can be autoclaved and stock solutions can be filter sterilized to avoid medium autofluorescence.
  • Cells are grown separately at 30°C and maintained in exponential phase overnight. Cells are washed twice with fresh medium and mixed in the different
  • Each cell mixture is loaded into the different cell loading wells of a microfluidic plate, such as a Y04 microfluidic plate (CellASIC, Hayward, CA) (FIG 1 IB).
  • a microfluidic plate such as a Y04 microfluidic plate (CellASIC, Hayward, CA) (FIG 1 IB).
  • Pressure forces cells to reach the chamber and remain trapped there.
  • 8 psi pressure (using a CellASIC microfluidic device) is applied in the cell wells to load them into the microfluidic chamber. Cells are retained in the chamber between the PDMS polymer and the bottom glass.
  • a constant flow of 5 psi is maintained during position selection.
  • the same input combination is introduced in each chamber at 5 psi. Once inputs are introduced (2-3minutes), pressure is reduced to 1 psi to minimize washing of the produced alpha factor.
  • the desired input combination is applied from the input wells and several positions of each chamber are imaged. Images are taken at regular intervals, such as every 10 minutes for 3 hours. After image processing, output is detected. For each position, the histogram of pixel intensities is calculated for the time 0 frame. An intensity threshold is defined around 95-99% of the total number of pixels. For the rest of time points, the average intensity of the pixels above the intensity threshold defined with time 0 is determined. Time course data is plotted for each chamber. Output is considered positive when there is a constant 5 or more fold increase above the threshold in any of the chambers.
  • Recombinant mammalian cells are constructed demonstrating that distributed biological computation can be extended to any cellular type.
  • cells are developed that respond to a particular stimuli to yield a controlled outcome, and cell-to-cell communication modules are developed that serve as the wiring on these systems.
  • the use of artificial intercellular communication networks in mammalian cells has previously been rather limited (e.g., Wang et al., 2001; Weber et al., 2007; Weber et al., 2009).
  • a basic circuit is designed with an AND gate establishing a circuit with two basic engineered mammalian cells.
  • the cells respond to two different stimuli, such as doxicycline and heavy metals, and use yeast pheromone (a-factor) as a wiring molecule (FIG. 17).
  • yeast pheromone a-factor
  • This scheme is similar to that used to implement an AND gate in yeast.
  • mCherry and vGFP have successfully been used to follow MAPK activation in HeLa cells by FACS and microscopy, indicating that similar readouts to those used in yeast can also be used in mammalian cells.
  • the yeast signal peptide a-factor encoded by the MF(a)l gene is fused to the Tet-On promoter that is tightly regulated by doxicycline.
  • the design of the second cell is based on Yin et al., 2005 which demonstrated that functional yeast G- protein-coupled receptor Ste2 can be successfully expressed in mammalian cells. When expressed in human HEK293 cells, Ste2 was shown to bind ⁇ -factor and to activate Erkl/2 mitogen-activated protein kinase. In addition, this cell has the ability to sense a second external input (heavy metals, e.g.
  • Zn +2 or Cd +2 controlling the expression of STE2 fused to the metallothionein (MT) promoter and to produce a final output (induction of GFP) under the control of an Erkl/2-responsive promoter such as FOS1 (under the control of the Elkl transcription factor).
  • This cell will be functioning as an AND gate responding to two stimuli (pheromone and heavy metals). The system is split into two cells to show that the wiring can be efficiently established to implement more complex circuits.
  • NOR negative-OR
  • NAND negative-AND
  • Other logic functions such as the NOR, OR or NAND gates can be implemented using different interconnected engineered mammalian cells by taking advantage of the TetON and TetOFF systems as well as the use of particular inhibitors of the ERK signaling pathway (e.g., PD98059).
  • the regulation of blood glucose levels is critical for complex organisms.
  • Glucose levels are primarily regulated by the balance of different hormones in humans; insulin and glucagon. Inappropriate regulation of those hormones results in glucose imbalances that can lead to severe diseases such as diabetes.
  • a multicellular circuit is developed that is able to regulate the balance between insulin and glucagon depending on glucose levels.
  • the input of the cellular circuit is the levels of glucose that are known to activate differentially a specific promoter encoding a glucose transporter ⁇ HXT1): Therefore, there are two possible states that are responded to: low levels of glucose( II), and high levels of glucose (1 ⁇ 2).
  • the multicellular circuit implementing this truth table is built using 3 different cellular types and one diffusible molecule as a wire.
  • This circuit can be implemented in yeast using the glucose transporters HXT1 (see FIG. 18).
  • RBS ribosome binding sites
  • the efficiency of the glucose-dependent transcriptional activation is modified, allowing for two different levels of activation, low and high.
  • Cell #1 responds upon low levels of glucose and produces a- factor.
  • FUS3 is expressed at high glucose levels under the HXTIuig h promoter.
  • the pheromone pathway is activated producing insulin.
  • Cell #3 express a transcriptional repressor (Inh), which inhibits the transcription of the glucagon gene expressed under the PInh promoter. Only if glucose levels are below the low threshold does the expression of the glucagon gene take place.
  • Inh transcriptional repressor
  • the same logic can be extended to human cells, for example by using the same glucose transporter HXT1.
  • the HXT1 regulatory element from S. cerevisiae is fused to the minimal cytomegalovirus promoter (HXT1-MIN) and inserted into an adenovirus for delivery to human fibroblasts, where it exhibits glucose-dependent transcriptional activation (Ferrer-Martinez, A., Riera, A., Jimenez-Chillaron, J.C., Herrero, P., Moreno, F., Gomez-Foix, A.M. (2004)
  • a glucose response element from the S. cerevisiae hexose transporter HXT1 gene is sensitive to glucose in human fibroblasts. J. Mol. Biol. 338(4):657-67)
  • Example 6 Population Control System Applied to Prevent Bacterial Contamination
  • cholera is a severe infection produced by the bacterium Vibrio cholerae and caused by ingestion of food or water contaminated. In third world countries cholera is still common.
  • a self-regulated multicellular control circuit able to detect and eventually destroy a pathogenic bacterium (e.g. Vibrio cholerae). For this, our system must behave as described below: i) The population of the artificial cellular circuit should be maintained at basal levels in the absence of pathogens in the medium,
  • the cellular circuit must respond in two different steps: 1) increasing its population, and 2) once a given population threshold is reached, the circuit will perform a specific action devoted to destroy the pathogen.
  • the population of the cellular circuit will be decreased to basal levels again.
  • the population levels of the circuit as well as the pathogen can be determined considering the levels of Acyl Homoserine Lactone (AHL) present in the medium.
  • AHL Acyl Homoserine Lactone
  • These molecules are secreted by bacteria as a part of the Quorum Sensing mechanism, being specific for each type of bacterium.
  • AHL Acyl Homoserine Lactone
  • the complex LuxR-AHL activates the transcription of genes expressed under the PL UX R promoter. Mutating the LuxR-like genes, it is possible to generate sensors that are activated by different AHL concentrations, i.e. sensing different bacteria concentrations in the medium.
  • cells in the circuit secrete a specific type of AHL as well as detect another specific AHL secreted by the pathogen. Furthermore, the cellular circuit population is self-regulated by the expression of a lethal protein under the control of another specific AHL (see below).
  • the pathogenic population can be reduced using different strategies.
  • cells of the multicellular circuit can produce bacteriophage viruses specifically mutated that can infect and destroy the pathogen but can not infect the cells of the multicellular circuit (see Avelar A. et al. 2009. "Phage-mediated bacterial bite back". iGem competition. MIT, USA.).
  • Other possibility would be the secretion of vesicles carrying a specific protein able to destroy the pathogen (see Kim J.Y. et al. Engineered bacterial outer membrane vesicles with enhanced functionality. 2008. J. Mol. Biol. 380(1): 51-66).
  • 1 1 1 1 0 1 C L and C H represent the activation of the sensor in a cellular circuit at two different concentrations in the medium (low and high, respectively).
  • A represents the concentration of pathogen that activates the response of the cellular circuit.
  • Kill C represents the expression of a lethal protein able to kill the control the cellular circuit.
  • Kill A represents the action against the pathogen, e.g. the synthesis of viruses or the secretion of vesicles.
  • FIG. 19 depicts an example of a multicellular circuit. All cells involved in the control circuit secrete N-3-(oxohexanoly)-HSL synthesized by Luxl and detected by LuxR. Cell #1 responds at low levels of N-3-(oxohexanoly)-HSL whereas Cell #2 has a mutated form of LuxR*, which is activated at high levels of N-3-(oxohexanoly)-HSL.
  • yeast X glucose
  • yeast Y maltose
  • the wine's pH is related with the malolactic fermentation process due to lactic bacteria present on grapes.
  • malolactic fermentation the transformation of malic acid into lactic acid takes place. Without the malolactic fermentation, it is not possible to obtain high quality wines.
  • One of the most limiting aspects of alcoholic fermentation is the balance of yeast and bacteria populations.
  • a multicellular circuit is designed that is devoted to control the specific yeast and lactic bacteria concentrations.
  • the yeast present on grapes are removed and that the fermentation is mainly carried out by modified yeasts:
  • fermentation is mainly executed by yeast X.
  • yeast Y population repressed.
  • the fermentation is carried out by yeast Y instead of yeast X (Y becomes dominant).
  • yeast population is below a determined upper threshold.
  • This circuit is implemented in yeast with a similar design to that used in Example 6 (FIG. 19).
  • Example 8 Implementation of a Memory Device
  • FIG 43A shows a schematic representation of a memory device that can store one bit of information.
  • the behavior of the one bit memory device is described in the truth table.
  • the state is indicated by the expression (1 logic) or lack of expression (0 logic) of a reporter protein GFP.
  • GFP* represents the next state of the system depending on the previous state of GFP and on the external inputs A and B. These external inputs allow writing 1 or 0 in the memory device.
  • the symbol x indicates that the previous state of the system is not relevant.
  • FIG. 43B shows a schematic representation of a circuit able to achieve signal restoration by using a bistable circuit
  • the bistable system changes the signal from the initial stable sate (low a and high b) towards the other stable state (high a and low b) generating an output production with a step-like shape (reporter protein GFP).
  • the output can be the input of the next logic block of a complex circuit ensuring circuit scalability.
  • the circuit upon linear input (A), the circuit produces a step-like output (GFP; a or b) that can be interpreted as a digital signal.
  • Example 9 Using inverted logic to reduce wiring requirements
  • the architecture of a circuit can be represented as an interconnected network of logic gates (nodes). These networks can be divided into three layers: the input layer that senses the external inputs, the output layer and the hidden layer that connects input and output layers (FIG. 44A).
  • An evolutive algorithm was used to explore the landscape of possible configurations of circuits able to implement any arbitrary Boolean function. This exploration was done imposing as criteria the minimization of the number of nodes and wires in the network and Distributed Computation.
  • FIG 44B shows an example of the pattern of connections in inverted logic circuits.
  • Each node in the input layer implements a 1- input 1 -output logic function, namely the Identity (where the state of the node is 1 only if the input is 1) or the NOT (where the state of the node is 1 only if the input is 0) functions.
  • the nodes of the output layer only implement the NOR function; meaning that only if all the signals coming from the input layer are 0 the output will be produced.
  • Boolean algebra any arbitrary Boolean function can be implemented using inverted logic. Furthermore, these circuits can be simplified using the standard rules of Boolean algebra. A theoretical framework is developed to demonstrate the potentiality of the use of inverse logic circuits. A user-friendly software interface for biological circuit design is developed where any user can introduce the desired computation and end up with
  • This algorithm includes a library of tools (using available databases of plasmids and other molecular items) helping the researcher to find the potential candidate (given the cell model chosen) for each interaction and regulation.
  • Boolean circuit implementation is in a mixed culture of different cellular types.
  • each node or logic gate can be implemented in a different cell type.
  • the connection between the cells forming the input layer and the cells forming the output layer can be implemented by using different diffusible molecules (e.g., a-factor from S. cerevisiae or a-factor from C. albicans).
  • all the diffusible molecules received by the same cell in the output layer can be identical.
  • arrows 1, 3 and 7 and/or arrows 2, 4 and 8 and/or arrows 5 and 6 can be implemented using the same diffusible molecule.
  • this circuit only requires three different molecules acting as wires. This allows for a simplification of the Boolean functions implemented in the output cells.
  • the cell only responds to a single input and hence the NOR function can be reduced to a NOT function (because NOT function is the particular implementation of the NOR function with a single input).
  • This method for circuit implementation takes advantage of the simplicity of the basic functions implemented by each cell (Id or NOT) making the cell engineering easier.
  • the number of required wires increases with the circuit complexity. Analysis of the correlation between the number of wires required with respect to the number of
  • a library of yeast cells is generated in which the reporter cells respond to the inverse logic (FIG. 14).
  • Input cells described for other embodiments herein can be reused to implement this approach.
  • the library of cells involves 2N cellular types since two different cells are responding with the Identity or the NOT functions for each different input.
  • pairs of cells are created that respond to different stimuli; each set of cells contains two different cells that respond to the same stimuli with a different logic. Therefore, pair-cells contain both identity (“Id”) and inverter ("In”) logic functions.
  • pair-cells are those responding to galactose; in the Id cell, the signal peptide a-factor encoded by MF(a)l gene is fused to the GALl promoter that is tightly regulated by a carbon source and it is strongly activated when galactose is present; the
  • NOT cell expresses the MF(a)l gene under the control of the HXT1 promoter which is inhibited by galactose. This pair of cells is regulated antagonistically by galactose and glucose (Regot et al., 2011).
  • Identity and inverter logic functions are represented as (Id) and (NOT) cells. Inputs are indicated in each pair of cells. All of the cells have the same output (a-factor). The universal reporter is inhibited by a- factor.
  • Inverse logic circuits together with the introduction of spatial constraints, which are discussed in Example 3, allow for the computing capacity of biological systems to increase greatly.
  • the biological circuits are constructed such that_only one extracellular wire is required and thus there is a clear reduction on the wiring problem in biological systems.
  • the cells are distributed in different compartments, such as by using chambers of microfluidics devices.
  • Each chamber contains the set of cells of the input layer that connects with the same cell of the output layer in the theoretical design. If a cell in the input layer is connected with more than one cell of the output layer, this cell will be present in more than one chamber.
  • FIG. 45 A demonstrates implementation with spatial separation (e.g., in microfluidics devices) of the circuit shown in FIG. 44B.
  • the physical separation between cells allows for a reduction of the number of wires to a single wire.
  • due to spatial segregation there will be no crosstalk induced by different wires and hence the same diffusible molecule can be used in every chamber of the microfluidics device.
  • an increase in circuit complexity can be associated with an increase in number of chambers required but not a corresponding increase in the amount of wiring required.
  • the cell that produces the output is always the same.
  • the library of engineered cells required can be fairly small and the elements used in building the internal molecular machinery can be re-used to obtain different cell types.
  • the maximum number of different cells responding to the external inputs is 2N and the maximum number of chambers is
  • FIG. 45C shows the correlation between the maximum number of required chambers and the maximum number of different functions that can be implemented with respect to the number of function inputs.
  • Example 10 Implementing reprogrammable cellular systems
  • FIG. 46 shows a schematic representation of a non-limiting example, wherein two different functions (described by the corresponding truth tables) can be implemented using the same cells with different distribution.
  • FIG. 46 presents a schematic diagram of the re-programmability in inverse logic circuits.
  • FIG. 46A shows the microfluidic implementation of a function described by the corresponding truth table (XOR function).
  • FIG. 46B shows the implementation of a different function, e.g., an IMPLIES function and corresponding truth table.
  • re-programming only requires relocating some cells in the compartments and removing one of them out of the system, but the cells do not need to be re-engineered.
  • cells 1 and 3 respond to input X 1 whereas cells 2 and 4 respond to input X 2 .
  • a more complex scenario for re-programmability is to have a complete library of cells within a device, e.g., each possible input cell implementing the Identity and the NOT function present in each chamber. With this device, any arbitrary function can be implemented. In order to implement a particular function in the SYNCOM chip, only some of the cells
  • FIG. 47 shows a schematic representation of this method of reprogrammability, through a non-limiting example of a general SYNCOM chip for functions with three inputs.
  • the whole library of engineered cells are present.
  • the activity of each cell can be inhibited separately by using specific inhibitors Ii.
  • the corresponding cells are not able to produce the diffusible molecule acting as a wire.
  • FIG. 47B demonstrates that upon introduction of a specific combination of inhibitors in each chamber, mixed with the inputs Xi, the circuit can be reprogrammed to implement a particular Boolean function.
  • FIG. 47C shows that the same device can be reprogrammed to implement a different Boolean function, modifying the combinations of inputs introduced in each chamber.
  • the general device shown in FIG. 47A acts as "hardware," whereas the specific combinations of inhibitors shown in FIG. 47B and FIG. 47C would act as "software.”
  • the behaviors of these circuits are described by the corresponding truth tables.
  • modified kinases in different signalling pathways As another non-limiting example, modified kinases in different signalling pathways
  • Example 11 Development of Distributed Computation circuits using isolated cells without extracellular wiring
  • spatial separation may not be possible.
  • multicellular distributed computation circuits are developed that do not involve spatial restrictions or wiring.
  • logic blocks of the circuit are organized into different cells without extracellular wiring.
  • Each cell is able to produce the output in a similar way to a microfluidics chamber.
  • An additional library of cells is created to build unwired consortia (2n cells are sufficient to implement all possible functions with n inputs. For example, with 8 cells, all functions with 3 inputs can be covered).
  • Each cell responds to two different sets of inputs: those that induce protein expression and those that prevent it by expressing a repressor.
  • the internal architecture of each cell is constant. When all inducing inputs are present and all repressor inputs are absent (the number varies depending on the implementation), a positive output will be produced. In some embodiments, inverted logic is not required. FIG.
  • FIG. 48 shows an example of implementation using three different cells. Further reduction techniques allow some cells not to respond to all inputs, further simplifying the design.
  • FIG. 48A depicts a schematic diagram of the general architecture of distributed computation circuits using isolated cells without extracellular wiring.
  • proteins Pi, P 2 and P 3 are expressed. These proteins can be expressed or repressed under different combinations of inducible promoters. For instance, X 1 and X 2 are activator inputs; hence, both inputs must be present simultaneously, whereas X 3 and X 4 act as inhibitory inputs expressing the repressor Inh, and must not be present for output production.
  • FIG. 48B shows a schematic representation of the implementation of the circuit shown in FIG. 44B. Arrows indicate activator inputs, whereas line-dot represents inhibitory inputs. Each cell will have similar internal architecture according to the scheme shown in FIG. 48A.
  • Library 1 Several non-limiting examples of libraries of engineered cells are developed. Library 1:
  • This library uses the yeast strain W303 (ade2-l his3-ll, 15 leu2-3, 112 trpl-1 ura3-l canl), which is genetically modified to be able to produce alpha factor from an inducible or repressible promoter. Strains are mfal and mfa.2 to prevent endogenous production of alpha factor. STE3 is deleted to avoid mating.
  • the library is extended by implementing cells connected through different wiring molecules. Modified a-factor from several yeast species (e.g., C. albicans, K. lactis and D. hansenii) are used together with their respective receptors.
  • This library contains yeast cells in which the reporter cells respond to inverse logic. It contains various pairs of cells that respond to the same stimuli with the identity (Id) or the NOT functions. Some of the input cells described herein for other applications can also be used for library development. All the pair-cells have the same output (such as a-factor) and the universal reporter is inhibited by a-factor following an inverse logic function.
  • inputs and engineered cells include: (1) Galactose; in the Id cell, the signal peptide ⁇ -factor encoded by MF(a)l gene is fused to the GALl promoter that is activated by galactose (Gasch et al., 2000); the NOT cell carries the MF(a)l gene under the control of the HXT1 promoter that is inhibited by galactose.
  • the signal peptide is controlled by the Tet-On (Id cell) and Tet-Off (NOT cell) regulated gene expression system (Belli et al., 1998), (3) NaCl; in the Id cell, the signal peptide is under the control of an osmoresponsive promoter such as STL1 (Posas et al., 2000; Causton et al., 2001); in the NOT cell, a system based on a transcriptional repressor responsive to osmostress is used.
  • MF(a)l gene is fused to some pheromone-repressed promoter such as PH081, FOX2 or QOR1 (Erdman et al., 1998; Spellman et al., 1998) then the cell will behave as an NOT cell.
  • some pheromone-repressed promoter such as PH081, FOX2 or QOR1
  • each cell responds to the whole set of inputs and is able to produce the output without the requirement of extracellular wiring.
  • the mating pathway is used and components are expressed or repressed under different input combinations.
  • Activating inputs such as NaCl or Gal induce proteins as the receptor for alpha-factor pheromone Ste2, the scaffold Ste5 or Ste7 MAPKK.
  • Inhibitory inputs induce expression of well-known
  • yeast repressors such as Skol or Tupl under the control of regulated promoters to control expression of other components of the mating pathway such as the MAPK Fus3.
  • the FUSl promoter fused to GFP is the output readout of this circuit.
  • XFP reporter gene such as GFP, RFP or others
  • FUSl promoter e.g. GFP, RFP or others
  • XFP is integrated in the FUSl ORF locus.
  • the ability of cells to respond to external stimuli is monitored by fluorescence in single cells (flow cytometry, such as using FACScalibur, Becton Dickinson, Franklin Lakes, NJ) and normalized to the maximal number of cells able to produce output signal.
  • Modified cells such as those in Libraries 1-3 are produced and are inhibited using small molecule inhibitors (Macia et al., 2009; Regot et al., 2011) to create reprogrammable circuits.
  • Small molecule inhibitors Macia et al., 2009; Regot et al., 2011
  • Each new engineered cell type is characterized by analysis of the Transfer Curve, meaning the cellular response with respect to different input levels.
  • an adequate transfer curve is characterized by several features (Weiss et al., 2003; Feng et al., 2004), such as i) a step-like shape, ii) linear or higher gain ranges in order to ensure that the signal will not be degraded from input to output for in a single cell, iii) adequate noise margins, without overlap between the high and the low state, and iv) each cell responds properly to the specific inputs and ignores the rest of inputs of the circuit.
  • Microscopy based microfluidic platform Cells are loaded into plates such as CellASIC (Hayward, CA) plates. Images are collected using NIS elements Software (Nikon, Melville, NY) and a Nikon Eclipse Ti Microscope and are analyzed with Matlab (Natick, MA). Implementing distributed computation in mammalian cells

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Abstract

Selon certains modes de réalisation, la présente invention concerne des circuits moléculaires multicellulaires qui présentent un calcul distribué.
PCT/EP2011/064083 2010-08-16 2011-08-16 Circuits synthétiques multicellulaires reprogrammables Ceased WO2012022741A1 (fr)

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