AI Article Synopsis

  • CFPS is gaining popularity in synthetic biology due to its advantages, and using linear DNA templates speeds up the process by removing cloning and extraction steps.
  • Linear DNA is prone to degradation by exonucleases present in cell extracts, but alternative strategies, like using exonuclease-deficient cells, can protect these templates without extra modifications.
  • The study details a method for preparing cell lysate from E. coli BL21 Rosetta2 ΔrecBCD, achieving protein expression levels similar to those obtained from plasmid DNA.

Article Abstract

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

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Source
http://dx.doi.org/10.3791/64236DOI Listing

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