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WO2019068006A1 - Lyse cellulaire contrôlée par stimulus - Google Patents

Lyse cellulaire contrôlée par stimulus Download PDF

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Publication number
WO2019068006A1
WO2019068006A1 PCT/US2018/053582 US2018053582W WO2019068006A1 WO 2019068006 A1 WO2019068006 A1 WO 2019068006A1 US 2018053582 W US2018053582 W US 2018053582W WO 2019068006 A1 WO2019068006 A1 WO 2019068006A1
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Prior art keywords
cell
protein
lysis
stimulus
cells
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Inventor
Kirsty A. MCFARLAND
Andrew P. MAGYAR
Ting PANG
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Charles Stark Draper Laboratory Inc
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Charles Stark Draper Laboratory Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • cells of interest can be genetically engineered/ programmed to lyse in response to a controlled, external stimulus, such as exposure to heat or light.
  • the methods described herein are biologically sensitive, for example, lysis of the cells with minimal effect on the biological activity of enzymes or other proteins of interest released from the lysed cell.
  • the present invention encompasses methods of genetically
  • transcription factor or “transcription regulator” defines a sequence-specific DNA-binding protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. Transcription regulators have functionality/activity to promote or block the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.
  • RNA polymerase the enzyme that performs the transcription of genetic information from DNA to RNA
  • the regulator can act as promoter or activator, (i.e., a protein that binds to an enhancer, or activator binding region of the DNA strand and enhances transcription from nearby promoter), or a repressor or blocker (i.e., a DNA binding protein that binds to the DNA strand and blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes).
  • promoter or activator i.e., a protein that binds to an enhancer, or activator binding region of the DNA strand and enhances transcription from nearby promoter
  • a repressor or blocker i.e., a DNA binding protein that binds to the DNA strand and blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes.
  • a cell lysis gene cassette comprising one, or more, cell lysis genes and one, or more, genetically modified transcriptional regulator genes associated with the expression of the cell lysis genes, wherein the transcriptional regulator encoded by the regulator gene(s) is stimulus- controlled.
  • the term "associated with” means that the expression of the cell lysis genes is regulated by, or otherwise dependent on, the biological activity or functionality of the transcriptional regulator protein.
  • the function/activity of the transcriptional regulator protein is, in turn, controlled by (directed by) the stimulus (i.e., upon exposure to the stimulus).
  • the transcriptional regulator gene can encode a repressor protein or an activator protein.
  • the cell lysis gene encodes a cell-disrupting protein (a lysis protein), or a biologically active fragment of the lysis protein such as a biologically active, lytic peptide.
  • a lysis protein a cell-disrupting protein
  • biologically active fragment of the lysis protein such as a biologically active, lytic peptide.
  • proteins are known to those of skill in the art and can include, for example, members of the holin, pinholin, spanin, or endolysin families of proteins, or other homologous proteins with the biological activity/functionality of disrupting cell membrane structure (i.e., permeabilizing the cell membrane).
  • the transcriptional regulator gene encodes a genetically modified protein that, once expressed, undergoes a conformational change in response to the stimulus, whereby the activity/function of the regulator is altered (e.g., the conformational change in the protein results in the expression of one, or more, cell lysis genes, or the repression of one, or more cell lysis genes, resulting in cell lysis/death.
  • a conformational change in the protein results in the expression of one, or more, cell lysis genes, or the repression of one, or more cell lysis genes, resulting in cell lysis/death.
  • Such stimulus can be, for example, changing the temperature during cell growth conditions or during analytical conditions.
  • the repressor protein AlpR has been modified (AlpR-A61T) to produce a thermally labile repressor protein.
  • the transcriptional regulator gene encodes a transcriptional regulator protein that is genetically modified to respond to an external stimulus such as light.
  • the transcriptional regulator gene can encode a transcriptional regulator protein that changes the conformation of, or activity of, the regulator protein upon exposure to a specific wave length of light, for example, via an azobenzene photo switch.
  • particles can be introduced into the medium surrounding the cells of interest, into the cells, or onto the cellular surface, that are stimulus-controlled, e.g., react to changes in heat or light exposure.
  • particles can be introduced into the cells that heat upon exposure to a specific energy source. The heat generated internally within the cell can trigger a conformational change of a regulator protein, resulting in the expression of the cell lysis genes and cell lysis.
  • compositions comprising a cell of interest, (e.g., an isolated cell) and the cell lysis gene cassette described above.
  • the cell can be a prokaryotic cell or a eukaryotic cell, and in particular is a bacterial cell.
  • the present invention comprises a transformed bacterial cell comprising the cell lysis gene cassette described herein.
  • the transformed bacterial cell can be an E. coli, or, more specifically, an E. coli strain BL21, and the cell lysis gene cassette can comprise one or both plasmid vectors pLysis or pINT HK.
  • the thermally labile variant of E e.g., an isolated cell
  • the cell lysis gene cassette described above.
  • the cell can be a prokaryotic cell or a eukaryotic cell, and in particular is a bacterial cell.
  • the present invention comprises a transformed bacterial cell comprising the cell lysis gene cassette described herein.
  • the transformed bacterial cell can be an E. coli, or, more
  • coli BL21 comprising recombinant plasmids pLysis and pINT HK is encompassed by the present invention.
  • a vector such as an expression vector comprising the cell lysis cassette (e.g., pLysis and/or pINT_HK) that is suitable for use in the methods of stimulus-controlled cell lysis described herein, and the cells described herein.
  • Suitable vectors can be designed by methods known to those of skill in the art.
  • Primer sequences as shown in FIG. 4C used to construct the plasmids are also encompassed by the present invention.
  • Methods encompassed herein include any method using the cell lysis cassette or vector described above to result in cell lysis or death.
  • a method of stimulus controlled cell lysis is encompassed by the present invention.
  • the method comprises the steps of introducing the cell lysis cassette or vector as described herein into the cell of interest.
  • Such methods of transforming or transfecting cells are known to those of skill in the art.
  • the cells are maintained under suitable conditions for growth or analysis.
  • the cells are exposed to the appropriate external stimulus, under conditions sufficient to alter the activity/function of the transcriptional regulator proteins encoded by the transcriptional regulator genes of the cassette, resulting in the expression of the cell lysis genes and cell lysis.
  • a further method encompassed herein is a method of ly sing cells wherein the cells are encapsulated within a droplet such as in a microfluidic device suitable and wherein the cells comprise a gene cassette as described herein.
  • a microfluidic device can be used for analysis of molecular interactions of proteins within cells of interest, or for protein synthesis.
  • Applications of droplet microfluidics can be found for example, in "Recent Advances in Applications of Droplet Microfluidics" Micromachines 2015, 6, 1249-1271.
  • the method can comprise specifically targeting and selectively lysing one, or more, cells in a droplet while the other cells in the droplet are not lysed and therefore remain viable for e.g., further molecular interactions for molecular pathway elucidation, signal detection or diagnostic purposes.
  • the method can comprise introducing a cell lysis gene cassette into a target cell and further substantially simultaneously, or subsequently, introducing a protein expression vector comprising a gene encoding a protein or biomolecule of interest into the target cell.
  • a cell such as a bacterial cell can be transformed with an expression vector comprising one, or more genes encoding one, or more, proteins/peptides/biomolecules of interest and co-transformed (or subsequently transformed) with a plasmid vector comprising the cell lysis gene cassette as described herein.
  • the protein expression vector can further comprise the cell lysis gene cassette.
  • the cell is incubated under conditions suitable for the expression of the protein of interest. After sufficient incubation, the cell lysis gene cassette is stimulated (e.g., by thermal stimulation of increasing the incubation temperature) and the targeted cell specifically lyses and releases the protein of interest into a cell lysate.
  • the cell lysate can be recovered by standard means and made available for further analysis or reactions.
  • the method can comprise recovering the cell lysate comprising the
  • proteins/biomolecules of interest i.e., cell lysate components
  • contacting the components of the cell lysate e.g., proteins
  • additional reagents or a second population of cells e.g., proteins
  • monitoring or detecting interactions of the cell lysate components e.g., cell lysate components
  • Reagents can be, for example, chemicals or molecules that detect a specific protein such as an enzyme or fluorescent detection reagent.
  • target cells can be selectively ly sed using the methods and cell lysis gene cassettes described herein.
  • a first population of targeted cells can be lysed and the cell lysate components can be maintained in contact with a second population of intact cells that, for example, express a protein or biomolecule that specifically interacts with the proteins in the cell lysate.
  • Such specific interactions can be, for example, a specific binding reaction such as between an antibody and antigen, or a cell surface receptor and ligand. These specific interactions can be detected by means known to those of skill in the art.
  • a further method encompassed by the present invention is a method of using gold nanoparticles to transduce an optical stimulus to a genetically controlled thermal stimulus for cell lysis.
  • the gold nanoparticles can be in solution, or selectively attached to the surface of the cell, or wherein the gold nanoparticles are encapsulated in the cells.
  • FIG 1 A A generalized he cell lysis cassette.
  • FIG IB the cell lysis cassette constructed for example 1 ⁇ 30 °C, repressor cl is expressed, repressing the transcription from pR promoter. Lysis genes are not transcripted either, due to the terminator sequence present upstream of pR' promoter. At > 42 °C, cl becomes inactive, anti-terminator Q is thus expressed. Q binds to the terminator sequence, unwinds it, so that lysis genes can be transcribed.
  • FIG 2A and 2B Amino acid sequences of the thermally labile repressor AlpR (SEQ ID NO. 1) and modified AlpR, AlpR-A61T (SEQ ID NO. 2).
  • FIG 3 A and 3B Plasmid maps of the two plasmids, pLysis (SEQ ID NO. 3) and PINT HK (SEQ ID NO. 4) used to produce a thermally labile E. coli BL21
  • FIG 4A-C shows a list of E. coli strains.
  • FIG 4B shows a list of plasmids.
  • FIG 4C is a list of primer sequences (SEQ ID NOS: 5-20) used in the assembly of the thermally labile E. coli BL21.
  • FIG 5 depicts different schemes for interacting gold nanoparticles (depicted as black circles) with E. coli to transduce optical signals to thermal signals to stimulate cell lysis.
  • FIG 6 is a schematic showing microfluidic work flow for encapsulation of cells in droplets and the selective thermal lysis of a subset of cells.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.
  • cells can be genetically manipulated to lyse in response to a controlled external stimulus using the molecular/genetic machinery (transcriptional and translational processes) of the host cell (e.g., cell of interest) by which the cell turns on/off the transcription of, or expression of, proteins essential to cell membrane integrity, resulting in cell lysis.
  • lysis can result from the initiation of a programmed cell death pathway such as apoptosis by inducing, promoting or repressing the transcription of certain genes associated with apoptosis.
  • Lysis can also result from the accumulation of cell-death associated proteins expressed at high levels within the cell, such as the accumulation of holins in bacteriophage-infected cells.
  • a genetic circuit see for example, "Principles of genetic circuit design", J. A. N. Brophy & C. A. Voigt; Nature Methods 11, 508-520 (2014) can be engineered into cells to enable cell lysis in response to a controlled stimulus.
  • the cells of interest are bacterial cells, but the methods described herein can be adapted to any cell, such as yeast or mammalian cells.
  • An example of the methods described herein is to engineer such a circuit based on the machinery of phages. This machinery includes the genes responsible for cell lysis and the transcriptional regulators that control this lysis.
  • the transcriptional regulator could be placed under the control of an inducible promoter or the regulator can be stimulus labile (thermal, optical, chemical).
  • the transcriptional regulator is a DNA binding protein that binds its cognate site as a dimer, and does not function except as at least a dimer. Therefore, anything that disrupts dimerization will also cause the cell lysis genes to be expressed.
  • Another method encompassed herein is to activate expression of cell lysis genes by placing them directly under the control of an inducible promoter, or activator or repressor protein.
  • the variant ⁇ bacteriophage holin SI 05 containing the A52G substitution is of particular interest, as its lysis occurs more rapidly after induction than the native SI 05 holin.
  • a gene cassette/genetic circuit can be designed that comprises one, or more, lysis genes and a thermal-labile repressor as described herein.
  • a genetic lysis circuit can be produced by placing the ASRRzRZl lysis cassette downstream from the pR' promoter, which is under repression by the thermally labile repressor, ACI857, expression of genes in the lysis cassette can be thermally induced.
  • Another example consists of the repressor AlpR, that could be rendered thermally labile by the A61T substitution, whose removal would allow expression of the lysis machinery AlpB and AlpC.
  • the sequence for ALP-R is shown in FIG. 2A and the modified sequence is shown in 2B.
  • the transcription factor AlpR represses expression of the AlpB-AlpC lysis proteins, which are native to Pseudomonas aeruginosa PAOl.
  • the genes encoding these proteins are introduced to E. coli, or another susceptible host, by introduction on a plasmid or integration in the host chromosome. After thermal upshift, the AlpR-A61T protein is no longer capable of maintaining repression of the lysis proteins, which causes the cells to lyse.
  • the expression of the lysis proteins may also be placed under control of other stimuli, for example, a chemical agent.
  • engineering the genetic circuit into a protein expression strain such as the E. coli BL21 can control cell lysis and enable the release of other expressed protein products.
  • Example 1 A thermally labile E. coli for protein expression
  • Fragment 2 containing the integrase from phage HK022 was amplified from plasmid pAH69, by primers F_pAH69 (SEQ ID NO: 7) and R_pAH69 (SEQ ID NO: 8).
  • the plasmid was transformed into NEB ® 10-beta Competent E. coli cells, and selected on LB plates containing 100 ⁇ g/ml ampicillin at 30 oC. The correct colony was named TL coli l.
  • the pLysis plasmid was constructed by four-way isothermal assembly.
  • Fragment 1 containing the attP site of phage HK022 was amplified from pAH144 using primers F_pAH144 (SEQ ID NO: 9) and R_pAH144 (SEQ ID NO: 10).
  • Fragment 2 containing the cl gene and pR promoter was amplified from plasmid pAH69 using primers F cIpR (SEQ ID NO: 11) and R cIpR (SEQ ID NO: 12).
  • Fragment 3 containing anti- terminator Q was amplified from lambda genomic DNA using primers F_Q (SEQ ID NO: 13) and R_Q (SEQ ID NO: 14).
  • Fragment 3 containing the lysis cassette was amplified from plasmid pS105 using primers F_pS105 (SEQ ID NO: 15) and R_pS 105 (SEQ ID NO: 16).
  • the plasmid was transformed into E.coli strain BW23473 and selected on LB plates containing 100 ⁇ g/ml spectinomycin at 30 °C.
  • the correct colony was named TL_coli_2.
  • TL_coli_l competent cells The pLy sis plasmid was then transformed into TL_coli_l competent cells, which was made by inducing the cells with ImM IPTG for 30 minutes to allow the phage HK022 integrase to express.
  • the transformants were selected on LB plates containing 35 ⁇ g/ml spectinomycin at 30 oC. Correct single integration event was confirmed by PCR amplification using primers CRIM HK Pl (SEQ ID NO: 17), CRIM P2 (SEQ ID NO: 18), CRIM P3 (SEQ ID NO: 19), and CRIM HK P4 (SEQ ID NO: 20).
  • the E.coli strain with pLysis integrated into the chromosome at HK022 attachment site (attB) was then named TL_coli_3.
  • phage Plvir was used to infect strain TL_coli_3.
  • the phage lysate was then used to infect BL21(DE3) by the standard transduction protocol.
  • the transductants were selected on LB plates containing 20mM Sodium Citrate and 35 ug/ml spectinomycin at 30 oC. Correct transductants were confirmed by PCR amplification using primers CRIM HK Pl, CRIM P2, CRIM P3, and CRIM HK P4, and renamed TL_coli_4.
  • a thermally labile Staphylococcus aureus [ 0038 ] Gram positive bacteria such as S. pyogenes, and S. aureus are known to be difficult to lyse by traditional means. Phage proteins of bacteria can be used to facilitate lysis of these organisms. More rapid, genetically-controlled lysis may facilitate the release and harvest of some bacterial products, such as RNA species that are sensitive to certain stimuli that are currently used to facilitate lysis by standard means.
  • Example of phage lytic proteins are the S. aureus bacteriophage vB SauS- philPLA88 derived endolysin LysH5 and fusion proteins between lysostaphin and a virion- associated peptidoglycan hydrolase HydH5, described in "The Phage Lytic Proteins from the Staphylococcus aureus," PLOS ONE, 2013 May, 8:5, e64671. Additional description of S. aureus phage lytic proteins is described in the review article, "Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?," mBio, 2018 Jan/Feb, 9: 1, e01923.
  • genes that produce these lytic proteins can be placed under the control of known elements that respond to certain stimuli, such as the thermally controlled repressors ACI857 or AlpR described above, or novel elements that may be derived from new sequences based on sequence homology.
  • the cell lysis gene(s) can be placed under control of light-controlled transcriptional regulator.
  • a photoactivatable regulator can also be referred to as a "photo caged", or “caged” regulator which is “uncaged” as a result of being exposed to a specific energy source/wavelength of light, and the caged transcriptional regulator is inhibited from activating gene expression (see, e.g., US Patent 7,541,193).
  • One method to control cell lysis is to engineer a transcriptional regulator as a DNA binding protein dimer for which dimerization is disrupted in response to an optical stimulus. This can potentially be achieved by the coupling of a photo switch molecule such as an azobenzene or a light responsive moiety to the dimer.
  • a regulator protein coupled to a photo switch can be produced using post translational modification methods known to those of skill in the art. (See e.g., Binder, et al, Interg. Biol. 2014, 6, 755; Mart, R.J. and Allerman, R. K., "Azobenzene photocontrol of peptides and proteins” Chem. Commun. 2016, 52, 12262).
  • Gold nanoparticles can be used extracellularly to raise the temperature of the surrounding cell media (FIG 5), can be functionalized to selectively interact with the cell surface to provide more localized temperature control, or can be utilized intracellularly.
  • the E. coli can be engineered to produce caveolin enabling endocytosis ("Constitutive formation of caveolae in a
  • Lysis of cells inside droplets can be important to enable the analysis of biomolecules that are contained within the cell.
  • Current approaches involve either the fusion of lysis reagents, electrical lysis, or the freezing and thawing of the droplets, all of which can disrupt the droplet, be denaturing, or otherwise interfere with this analysis.
  • the ability to control the lysis genetically in response to a particular signal will enable the selection of a broad range of stimuli. In particular, the use of a thermal or optical stimulus, as described above, will be non-invasive to the droplet.
  • a mixed population of cells can be contained within a droplet and some portion of this population could contain genetic circuits enabling them to be selectively lysed.
  • An example application of this is to have a biomolecule-producing bacterium that is co- encapsulated in the same droplet as a different type of cell. The selective lysis of the biomolecule-producing cell would enable interactions of the biomolecule with the second cell type, which could then be assayed, e.g., in high-throughput screening analysis.
  • ⁇ Encapsulator System Dolomite Bio
  • Dolomite Bio can enable the encapsulation of individual cells in pL aqueous droplets dispersed in an oil phase.
  • Cells containing the machinery for thermal cell lysis and that produce a protein, antibody or other compound of interest are prepared in one set of droplets and a second set of cells that do not lyse thermally are prepared in a second set of droplets.
  • a microfluidic chip is used to merge droplet set 1 with droplet set 2, creating droplets that contains a cell that will lyse under a thermal stimulus and cells that will not lyse under a thermal stimulus.
  • the droplets are heated to 42 °C causing the thermally responsive cells to lyse (FIG.
  • the cell lysate containing any protein or other products will interact with the cells from droplet set 2 and this response can be monitored.
  • FADS fluorescent activated droplet sorting
  • the cells that thermally lyse can also contain gold nanoparticles and a light source can be used to selectively lyse cells rather than a heat source.

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Abstract

L'invention concerne des compositions de lyse cellulaire contrôlée par stimulus et des procédés d'utilisation de ces compositions.
PCT/US2018/053582 2017-09-29 2018-09-28 Lyse cellulaire contrôlée par stimulus Ceased WO2019068006A1 (fr)

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Cited By (7)

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CN111909918A (zh) * 2019-05-10 2020-11-10 中国科学院微生物研究所 一种内溶素突变体及其编码基因与应用
CN113856778A (zh) * 2021-11-03 2021-12-31 清华大学 一种选择性检测微生物的微流控芯片、装置和方法
EP3914692A4 (fr) * 2019-01-25 2022-11-02 The Australian National University Cellules encapsulées
WO2023015440A1 (fr) * 2021-08-10 2023-02-16 中国科学院深圳先进技术研究院 Bactéries obtenues par génie génétique pour une thérapie antitumorale basée sur un biofilm bactérien, procédé de construction correspondant et application de celui-ci
WO2023015441A1 (fr) * 2021-08-10 2023-02-16 中国科学院深圳先进技术研究院 Bactérie d'ingénierie de lyse à commande par la lumière, procédé de construction correspondant et son utilisation
CN115704041A (zh) * 2021-08-10 2023-02-17 中国科学院深圳先进技术研究院 一种光控裂解工程菌及其构建方法和应用
CN115704006A (zh) * 2021-08-10 2023-02-17 中国科学院深圳先进技术研究院 基于细菌生物被膜的肿瘤治疗工程菌及其构建方法和应用

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