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| The Homemade E. coli S30 extract was prepared using E. coli strain BL21 (DE3) with loss of function mutations in OmpT endoproteinase and lon protease genes. This E. coli strain can express T7 RNA polymerase under the control of LacI, which is inducible by IPTG 1.
The simple constitutive gene expression device BBa_T9002 has been used to characterize this cell-free chassis
||Flow chart showing the protocol for making homemade E. coli S30 extract. The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al 1.
|| Graph 1. Graph of fluorescence (au) against time (minutes). The pink line represents the negative control consisting of homemade E. coli S30 extract and homemade premix with no DNA; the light blue line represents the reaction using homemade E. coli S30 extract and homemade premix with DNA; the yellow line represents the reaction using commercial E. coli S30 extract and commercial premix with DNA; the black line represents the reaction using homemade E. coli S30 extract and commercial premix with DNA; the green line represents the positive control consisting of GFP protein in solution. The inducible gene expression device BBa_T9002has been used.
- 1 pmid=16797767