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Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis.

Logan R Burrington1,2, Katharine R Watts1,2, Javin P Oza1,2

  • 1Chemistry and Biochemistry Department, California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, California 93407, United States.

ACS Synthetic Biology
|July 16, 2021
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Summary

Cell-free protein synthesis (CFPS) reaction times were significantly reduced by optimizing extract composition and additives. This advance makes CFPS faster for biomanufacturing, education, and diagnostics.

Keywords:
CFAIcell-free protein synthesisin vitro transcription/translationrapidratesynthetic biology

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Cell-free protein synthesis (CFPS) is a versatile platform for on-demand protein production.
  • Current CFPS applications are often limited by lengthy reaction times, despite improvements in yield.

Purpose of the Study:

  • To establish benchmark reaction times for CFPS using E. coli extracts and commercial kits.
  • To optimize in-house CFPS systems to significantly reduce reaction times for various applications.

Main Methods:

  • Benchmarking reaction times for CFPS across different E. coli extracts.
  • Optimizing reaction composition and titrating low-cost additives in in-house CFPS systems.

Main Results:

  • Reaction times were reduced by 30-50% for biomanufacturing applications, achieving high protein titers.
  • Reaction times were reduced by over 50% for education and diagnostics, reaching the sfGFP detection limit.
  • Visible sfGFP signal was observed in under 10 minutes under optimized conditions.

Conclusions:

  • Optimized CFPS formulations enhance reaction speed without compromising protein yield.
  • Reduced reaction times expand the utility of CFPS for rapid biomanufacturing, education, and diagnostics.
  • CFPS is a more accessible and user-defined platform with these time enhancements.