E. coli S30 Extract System for Linear Templates Technical Bulletin

Literature # TB102

The E. coli S30 Extract System for Linear Templates, prepared using minor modifications of the protocol described by Lesley and colleagues, allows successful transcription/translation of linear DNA templates. The investigator only needs to provide linear DNA containing a prokaryotic E. coli-like promoter and sequence for a ribosomal binding site. In vitro-generated RNA from DNA templates lacking an E. coli promoter also may be used in this system, but protein yields will be decreased to 1–10% of that produced from linear DNA templates.

The S30 Extract for Linear Templates is prepared from an E. coli B strain (SL119), which is deficient in OmpT endoproteinase, lon protease and exonuclease V (recBCD). The absence of protease activity results in greater stability of expressed proteins. The recD mutation of the SL119 strain produces a more active S30 Extract for Linear DNA than the previously described nuclease-deficient, recBC-derived S30 extracts. However, the S30 Extract for Linear Templates is less active than the S30 Extract System for Circular DNA (Cat.# L1020).

An easy-to-perform, non-radioactive positive control reaction using the Luciferase Assay Reagent provided allows detection of luciferase gene expression in the E. coli S30 System for Linear Templates. The control reaction produces high light output for several minutes, allowing the researcher to choose from several methods of detection, including simple visual observation of luminescence.