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WO2016048994A3 - Systems and methods of cell-free protein synthesis in droplets and other compartments - Google Patents

Systems and methods of cell-free protein synthesis in droplets and other compartments Download PDF

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Publication number
WO2016048994A3
WO2016048994A3 PCT/US2015/051401 US2015051401W WO2016048994A3 WO 2016048994 A3 WO2016048994 A3 WO 2016048994A3 US 2015051401 W US2015051401 W US 2015051401W WO 2016048994 A3 WO2016048994 A3 WO 2016048994A3
Authority
WO
WIPO (PCT)
Prior art keywords
protein
droplets
cell
free
protein synthesis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2015/051401
Other languages
French (fr)
Other versions
WO2016048994A2 (en
Inventor
David A. Weitz
Shaorong Chong
Huidan ZHANG
Nai Wen CUI
Ye Tao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
New England Biolabs Inc
Harvard University
Original Assignee
New England Biolabs Inc
Harvard University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New England Biolabs Inc, Harvard University filed Critical New England Biolabs Inc
Publication of WO2016048994A2 publication Critical patent/WO2016048994A2/en
Publication of WO2016048994A3 publication Critical patent/WO2016048994A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention generally relates to cell-free protein synthesis in microfluidic droplets. Droplets may be used to encapsulate genetic information (DNA/RNA), and through cell-free protein synthesis, provide a linkage of the genetic information with the functional information, e.g., the activity of the expressed protein, and used to convert the functional information into a detectable signal, e.g., to allow sorting of the droplets and retrieve genetic information associated with the drops. In one set of embodiments, microfluidic droplets containing a cell-free protein synthesis system designed to detect protein-protein interaction (e.g., in vitro two-hybrid systems or IVT2H) can be used for high-throughput screening, e.g., of protein binders. This drop-based two-hybrid system may include two (or more) fusion proteins that can bind to each other such that their binding produces a complex that is able to produce a nucleic acid. The nucleic acid may be expressed to produce a protein. In certain embodiments, the protein may be produced within a cell-free system. The protein may be fluorescent or otherwise determinable, such that determination of the protein may be used to allow assays to occur within the droplets, to allow sorting of the droplets to occur, or the like, e.g., as discussed below, for instance, for screening or other applications.
PCT/US2015/051401 2014-09-23 2015-09-22 Systems and methods of cell-free protein synthesis in droplets and other compartments Ceased WO2016048994A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462054263P 2014-09-23 2014-09-23
US62/054,263 2014-09-23

Publications (2)

Publication Number Publication Date
WO2016048994A2 WO2016048994A2 (en) 2016-03-31
WO2016048994A3 true WO2016048994A3 (en) 2016-05-26

Family

ID=55582238

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/051401 Ceased WO2016048994A2 (en) 2014-09-23 2015-09-22 Systems and methods of cell-free protein synthesis in droplets and other compartments

Country Status (1)

Country Link
WO (1) WO2016048994A2 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018067792A1 (en) * 2016-10-07 2018-04-12 President And Fellows Of Harvard College Sequencing of bacteria or other species
WO2020077274A1 (en) 2018-10-11 2020-04-16 Berkeley Lights, Inc. Systems and methods for identification of optimized protein production and kits therefor
EP3828283A1 (en) * 2019-11-28 2021-06-02 Diagenode S.A. An improved sequencing method and kit
US11788137B2 (en) 2019-09-30 2023-10-17 Diagenode S.A. Diagnostic and/or sequencing method and kit
AU2021327851B2 (en) * 2020-08-21 2025-07-10 Nuclera Ltd Monitoring of in vitro protein synthesis
GB202311993D0 (en) * 2023-08-04 2023-09-20 Cambridge Entpr Ltd Method and system
KR20250074706A (en) * 2023-11-15 2025-05-28 대한민국(방위사업청장) Method to detect target molecules based on cell-free protein synthesis and retroreflection signal

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030008398A1 (en) * 1996-12-11 2003-01-09 Rolf Mueller Self-enhancing, pharmacologically controllable expression systems
US20090304699A1 (en) * 2006-05-24 2009-12-10 Biogen Idec Ma Inc. Methods of treating fibrosis
WO2012139134A2 (en) * 2011-04-07 2012-10-11 Coferon, Inc. Methods of modulating oncogenic fusion proteins
US20130171328A1 (en) * 2010-06-02 2013-07-04 Ganesh M. Kishore Production of steviol glycosides in microorganisms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030008398A1 (en) * 1996-12-11 2003-01-09 Rolf Mueller Self-enhancing, pharmacologically controllable expression systems
US20090304699A1 (en) * 2006-05-24 2009-12-10 Biogen Idec Ma Inc. Methods of treating fibrosis
US20130171328A1 (en) * 2010-06-02 2013-07-04 Ganesh M. Kishore Production of steviol glycosides in microorganisms
WO2012139134A2 (en) * 2011-04-07 2012-10-11 Coferon, Inc. Methods of modulating oncogenic fusion proteins

Also Published As

Publication number Publication date
WO2016048994A2 (en) 2016-03-31

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