As RNAi is an eukaryote-specific regulatory mechanism, a proof of principle in prokaryotes had to consist of an RNA-based workaround. For E. coli, we decided to implement a regulatory system based on short transcriptional activating RNAs (STARs) that can function as RNA-logic regulators and achieve up to 94-fold activation of reporter genes [1]. Therefore, we designed a two-part plasmid system that functions as activator and target.
The Attacker Plasmid
The first plasmid synchronises the production of anti-sense STAR mRNA to the transcription of an RFP that functions as a reporter gene and stands in for the plant-specific reaction gene. In this plasmid, controlled by an inducible pBAD promoter the RFP is followed by the anti-sense STAR-sequence. STAR is flanked upstream by a Hammerhead Ribozyme and downstream by a Hepatitis Delta Virus Ribozyme, as well as a BBa_B0012 terminator. We use the anti-sense sequence AD1.A5 that has shown up to 94-fold activation properties in Chappel J. et al. 2015. When transcribed to mRNA, the ribozymes will fold into their enzymatic secondary structure and cleave the anti-sense STAR [2]. As a result, the anti-sense STAR is able to function independently as if transcribed separately and can sequester to its complementary sense-sequence on the target plasmid.
The Target Plasmid
The target plasmid consists of a constitutive BBa_J23119 promoter, followed by a terminator sequence and a reporter amilGFP. The terminator sequence is again taken from Chappel J. et al 2015 (AD.S5). Part of the 5’-start of the terminator sequence is complementary to the anti-sense STAR sequence. As a result, the cleaved anti-sense STAR will sequester the complementary mRNA sequence of the terminator and thereby negate the terminator’s hairpin structure. This enables the transcription of the reporter amilGFP.
The Ribozymes
The Hammerhead ribozyme and Hepatitis Delta Virus ribozyme are both cis-cleaving ribozymes with their cleavage region positioned in a way that guarantees a precise cut of STAR sequence. This creates a homogenous sRNA that could not be achieved with a regular expression of STAR via separate promoter and terminator.
With this construct, we aim to show that the regulatory STAR-mechanism can be synchronised to the expression of a gene of interest. The ribozyme-based cleavage mechanism can release the synchronised mRNA effectively into the cell where it can function independently of the gene of interest.
