Contribution

E. coli Promoter - yqgW
E. coli Promoter - rho
Existing Part Contribution

This year the team decided that we could make a useful contribution to the iGEM community though the identification of new Bacillus subtilis (B. subtilis) promoter sequences. This experiment allowed us to identify new B. subtilis promoters with the potential to express our enzymes. In addition, our team contributed data to the existing part, AmilCP (BBa_K592009).

Promoters

Previously our supervisory team had identified 30 putative constitutive promoters from the genome of B. subtilis  168. They asked us if we wanted to investigate whether any of them were active and if so, if we wanted to use them in our project. Of course, we said yes! These putative constitutive promoter sequences were found through identifying 100 bp sequences located upstream of every coding sequence from the B. subtilis 168 genome. Sequences that overlapped with other coding sequences or those containing transcription factor binding sites were discarded. 30 of these 250 bp long sequences were then randomly selected and synthesised. Using TypeIIs cloning we inserted the promoter upstream of the coding sequence for either AmilCP (blue chromoprotein) or MeffCP (pink chromoprotein). This allowed us to visually identify which sequences contained active promoters through the detection of blue and red/purple respective colour changes.  This provided us with 60 constructs in total which we initially tested in a cell free protein expression system (based on B. subtilis WB800N). The use of a cell free protein expression system allowed the team to conduct this experiment off campus. This meant the team could collect data during the period where we did not have lab-access due to COVID-19 imposed campus closures. Through spectrophotometry readings we could identify which DNA sequences contained B. subtilis promoter sequences that were active in the cell free environment. No colour change was observed for any of the constructs using the cell free method indicating no protein expression. When we were finally able to get into the lab, the plasmids were transformed into E. coli  DH5 alpha. From these transformations two promoters were found to be active in E. coli ; yqgW (K3371000) and rho (K3371001) or as we know them, promoters 30 and 15!

E. coli Promoter - yqgW

View this part on the Registry

This promoter was found upstream of an open reading frame annotated as yqgW (a conserved protein of unknown function) in the published genome sequence of B. subtilis 168 (NC_000964). After 24 hours, growth colonies containing the AmilCP gene appeared blue (Figure 1), indicating that despite the promoter originating from  B. subtilis, the promoter was active in  E. coli.

Figure 1. E. coli  DH5 colonies transformed with promoter 30 from B. subtilis 168 containing AmilCP gene after 24 h

E. coli Promoter - rho

View this part on the Registry

This is a constitutive promoter identified from promoter mining of the B. subtilis 168 genome. After 24 h growth colonies containing the MeffCP gene appeared pink (Figure 2), indicating that despite the promoter originating from  B. subtilis, the promoter was active in  E. coli.

Figure 2. E. coli  DH5 colonies transformed with promoter 15 from B. subtilis 168 containing MeffCP gene after 24 h

Unfortunately, due to limited access to the lab we were unable to investigate whether the two promoter’s were active in  B.subtilis. We have however contributed two new constitutive  E. coli  promoters to the registry and we hope that future teams will be able to make use of this putative promoter in  B.subtilis.

Existing Part Contribution

We have investigated the expression of AmilCP under control of two putative constitutive promoters identified in the genome of B. subtilis 168. Details about these promoters can be found under the parts page BBa K3371000 and BBa K3371001. After transformation of E. coli DH5a with plasmids containing the AmilCP CDS under the control of our two promoters, we initially confirmed expression of AmilCP as our colonies appeared blue! We picked three blue colonies from each of the transformation plates and used them to inoculate E. coli cultures (in LB broth). We monitored growth of the cultures (OD 600nm, data not shown) and expression of the protein (A 420 nm, blue light region of the electromagnetic spectrum) over 24 h.

The graph show that our promoters drive expression of AmilCP at different times during growth. With our promoter 30 BBa K3371000 there is a ~3 h lag phase in expression and maximum expression occurs after 10 h. With our promoter 15 BBa K3371001, there is no lag phase and maximum expression occurs earlier 7-8 h. Unfortunately due to restricted time available in the lab we were unable to investigate further as to why this might be the case.

References