Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements

作者：Kelwick Richard; Webb Alexander J; MacDonald James T; Freemont Paul S

来源：Metabolic Engineering, 2016, 38: 370-381.

DOI：10.1016/j.ymben.2016.09.008

• Synthetic biology
• Bacillus subtilis
• Cell-free transcription-translation
• Regulatory element prototyping
• Promoter library
• Luciferase assay

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8 mu M GFP, which gave a similar to 72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P-grac (sigma A) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

• 出版日期2016-11