Difference between revisions of "Team:Nottingham/Improve"

Improved parts

We have improved three promoters from the iGEM registry by adding a consensus ribosomal binding site (RBS) from the thiolase gene of Clostridium acetobutylicum to each, which functions in Gram-positive and Gram-negative bacteria. We then performed a robust characterisation through a GFP assay using a spectrophotometer in conjunction with calibration curves generated during the iGEM Interlab study, and expressed their strengths in standardised units of fluorescence.

Original promoters from iGEM registry:

Original promoters with RBS (Ribosomal binding site):

Additionally we have added four new promoters to the iGEM registry with the same ribosomal binding site from the thiolase gene of C. acetobutylicum. The four promoters are from C. acetobutylicum (Pcac_thl) [Bba_K2715010], Clostridium sporogenes (PCsp_fdx) [Bba_K2715011] and two from Clostridium difficile (PCdi_TcdA) [Bba_K2715012] and (PCdi_TcdB) [Bba_K2715013].

We characterized all seven promoters in both E.coli and C. difficile. C. difficile is a Gram-positive anaerobic organism with significant differences to the E. coli chassis for which existing characterisation was performed. The existing registry promoters BBa_J23114, BBa_J23106, and BBa_J23119 were characterised for expression strength using a GusA assay in C. difficile. The four novel registry parts were characterised alongside the existing registry promoters in a GFP assay in E. coli as well as in a GusA assay in C. difficile. The most remarkable conclusion from the E. coli GFP assay of these promoters is that both of the suspected strong C. difficile promoter PCsp_fdx and Pcac_thl were stronger than any of the three existing registry promoters we assayed; with Pcac_thl producing around three times the concentration of fluorescein (0.3235µM) as the positive control used in the InterLab studies (0.0958µM).

As part of our collaborative studies, we invited two other iGEM teams (Imperial College London and University of Warwick) to characterise our composite parts using their equipment and calibration curves, and the observed reproducibility between laboratories further validated our observations. Furthermore this part has now been characterised in a Gram-negative and a Gram-positive non-model organism. When tested in the Gram-positive organism it was driving expression of gusA.

Our main objective in characterising these promoters was to find a suitable pair of strong promoters to use in our subsequent dCas9 or asRNA projects. For this the GusA assay within C. difficile was most relevant since this is the chassis in which these constructs would be acting. The C. difficile GusA assay clearly showed that none of the three existing registry promoters from E. coli had any detectable activity in C. difficile. By far the strongest promoter we were able to measure was PCsp_fdx which was around 7.5 times stronger than the next strongest promoter we found (PCdi_TcdA). We were unable to clone the strongest promoter from the E. coli GFP assay PCac_thl into a GusA reporter plasmid. This is likely because of the toxicity of the gusA gene in E. coli and since we know that PCac_thl is the strongest of our promoters in E. coli it is unsurprising that this was the most problematic plasmid to clone. As a result we did not measure the strength of PCac_thl in C. difficile, but due to its measured strength in C. difficile as well as its widespread use for overexpression studies in Clostridia we decided to select it alongside PCsp_fdx as a promoter to use in the next stage of our project.

GFP assays. The graphs above represent the data accumulated from the GFP assay reproduced at Imperial College London and Warwick University. They all show similar levels of fluorescence.