An intronic, self-splicing riboswitch is configured for enzyme-product specificity by using an appropriate aptamer. The riboswitch is used in a sensing-expression construct in a transformed cell, whereby the presence of an enzyme product in the cell triggers self- splicing of the intron sequence to restore the reading frame of the reporter gene. This then drives expression of the gene product. The sensing construct expresses a protein which marks the cell or permits its growth or survival in or on an otherwise selective media. In this way, introduction or the presence of such product sensing-reporter constructs in cells can be harnessed to provide a multi-parallel rapid screening of cells or libraries for desirable enzyme variants. A "reporter cascade" is set up for GFPuv production. The T7 polymerase gene is carried on the bacterial genome. The enzyme's functionality depends on maturation (RNA splicing that restores open reading frame), which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method.
|Publication status||Published - 20 Oct 2016|