We developed a composite part (BBa_K1987000) to facilitate the testing of promoter/RBS combinations such as the ones featured in our project. Most of the part’s sequence is identical to other GFP reporter systems: it contains a promoter and RBS, followed by sequence coding for GFP with an attached degradation tag. Flanking the promoter and RBS, however, are BSAI sites that allow for the insertion of any other promoter/RBS pair via Golden Gate assembly. Users simply add the right tails to the forward and reverse primers which anneal to their promoter/RBS and then complete a routine PCR (Forward primer tail: 5’- AGGTCTCATAGA - 3’. Reverse primer tail: 5’ - TGGTCTCTCGCAT - 3’). This PCR product can then be used in a Golden Gate reaction, where it is swapped into the correct spot in the plasmid. The fluorescence produced by a bacterial culture containing this plasmid can then be measured over time in order to characterize the promoter/RBS activity.
The main advantage offered by our measurement system over others is that it allows for seamless insertion. Unlike many enzymes used for traditional cloning, BSAI cleaves DNA outside of the recognition site. This means a scar will not be left from the recognition site post-assembly. Scarless assembly is important in this application because RBS strength can depend strongly on its distance from the start codon of the following gene. Without this scar, promoter/RBS combinations can be more accurately characterized. A secondary advantage of our system is the ease of use. It requires only a PCR reaction followed by the single-step Golden Gate reaction to insert any promoter/RBS combination into the construct. Once inserted, characterization can be completed through any desired GFP assay.