Oct 3, 2013

Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology

Journal of Visualized Experiments : JoVE
Zachary Z SunVincent Noireaux

Abstract

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA pol...Continue Reading

  • References27
  • Citations22

References

  • References27
  • Citations22

Citations

Mentioned in this Paper

Buffers
Alkalescens-Dispar Group
Viral Proteins
Transcription, Genetic
Proteins, Recombinant DNA
Promoter
Transcriptase
Protein Biosynthesis
Escherichia coli
Synthetic Biology

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