Team:Exeter/Parts
Parts
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Part:BBa_K3371000
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Bacillus subtilis 168 Promoter (yqgW)
Constitutive promoter identified from promoter mining of the Bacillus subtilis 168 genome.
Usage and Biology
Our team is developing a novel precipitation method using bacteria engineered with enzymes capable of producing the carbonate ions required for precipitation of calcium carbonate. Our chosen bacterial chassis is Bacillus subtilis due to the species natural ability to tolerate changes in pH [1]. 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 and of course we said yes! 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). We know it as promoter 30!
Characterisation
Using TypeIIs cloning we inserted the promoter upstream of the coding sequence for either AmilCP (blue chromoprotein) or MeffCP (pink chromoprotein) and transformed the resulting plasmids into E. coli DH5 alpha. After 24 h 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
Unfortunately due to the COVID-19 pandemic and limited access to the lab we were unable to investigate whether the promoter was active in B. subtilis. We have added a constitutive E. coli promoter to the registry and we hope that future teams will be able to make use of this putative promoter in B. subtilis.
Part:BBa_K3371001
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Bacillus subtilis 168 Promoter (rho)
Constitutive promoter identified from promoter mining of the Bacillus subtilis 168 genome.
Usage and Biology
Our team is developing a novel precipitation method using bacteria engineered with enzymes capable of producing the carbonate ions required for precipitation of calcium carbonate. Our chosen bacterial chassis is Bacillus subtilis due to the species natural ability to tolerate changes in pH [1]. 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 and of course we said yes! This promoter was found upstream of an open reading frame annotated as rho (transcriptional terminator) in the published genome sequence of B. subtilis 168 (NC_000964). We know it as promoter 15!
Characterisation
Using TypeIIs cloning we inserted the promoter upstream of the coding sequence for either AmilCP (blue chromoprotein) or MeffCP (pink chromoprotein) and transformed the resulting plasmids into E. coli DH5 alpha. 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
Unfortunately due to the COVID-19 pandemic and limited access to the lab we were unable to investigate whether the promoter was active in B. subtilis. We have added a constitutive E. coli promoter to the registry and we hope that future teams will be able to make use of this putative promoter in B. subtilis.
Part:BBa_K592009
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AmilCP, blue chromoprotein
"This chromoprotein from the coral Acropora millepora, amilCP, naturally exhibits strong color when expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation." - Uppsala-Sweden iGEM team, 2011.
Our 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_K3371000, 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.
Ordered Parts
We used TypeIIs cloning to build constructs with the coding sequences for our chosen enzymes and a variety of promoters, RBS and terminators. The team built two sets of constructs. One set was designed for protein expression in E.coli BL21(DE3) while the second set was designed for protein expression in our terminal chassis, B. subtilis WB800N.
Although we were able to spend time in the lab building our constructs, we were not able to access the lab often enough to be able to test them.
References
[1] Wilks et al. (2009) Applied and Environmental Microbiology. 75 (981)
