Abstract:
Cell free extract (CFE) being an open system, provides a plethora of opportunities like prototyping of genetic circuits, non-canonical amino acid incorporation and high throughput analysis which cannot be performed using intact cells. From the early sixties researchers are utilizing the CFE system for various applications. Targeted high yield protein production being one of the most demanding applications of them all. Expression of toxins can also be achieved in a cell free extract system which is impossible in intact cells. Genetic switches can be designed in such a way that can detect viral RNA when linked to a reporter system. Cell free extract is an excellent platform to express these synthetic gene circuits. Our aim here is to develop and optimize a cell free extract system which would be capable of expressing a synthetic gene circuit. The easiest way to detect expression is to express a fluorescent proteins. Reporter proteins like GFP and variants are prime candidates for such reporter systems. We expressed a monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP) in Escherichia coli DH5 alpha and BL21 (DE3) cells and prepared an extract of them. We wanted to add plasmids that express the genes encoding these proteins in our homemade extract. To this end we needed to establish the stability of expression of these reporter proteins in the extract system. We found out that the iGEM repository supplied mRFP expressing plasmid was constitutive.Both GFP and mRFP were found to be stable in the exact system. We then made our home made cell free extract and optimized conditions for expression. A GFP expression plasmid was added to the homemade extract system and expression levels comparable to cells transformed with the plasmid were observed. We also constructed a LacZ expression plasmid and expressed it in homemade extractsystem.We find that our homemade E. coliBL21 extract with energy-mix is capable of transcription and translation. In future this would may become part of a larger project to deploy such TxTr systems for viral diagnostics.
