Team:Guelph/Contribution
For its 2019 contribution to parts previously held on the iGEM Registry, iGEM Guelph characterized the expression of BBA_K1349002 at different temperatures in the hopes of adding more data to this novel form of AmilCP.
Given the instability of BBa_K1343022, the source of amilCP in BBa_K3189015, we also used BBa_K1349002 - a version of amilCP codon optimized for expression in E. coli by iGEM Uppsala in 2018 - to combine with the BBa_K3189001 promoter and create a tetracycline biosensor with E. coli optimized AmilCP, submitted to the registry as BBa_K3189014, using the same molecular cloning procedures as for BBa_K3189015 as outlined in the Experiments section of our Wiki.The identity of the part was confirmed by Sanger sequencing.
From these results it appears that 24 hours at 25°C produced a stronger visual colour change when compared to colonies grown at 37°C for the same amount of time. This is most likely a result of reduced expression or stability of AmilCP at higher temperatures. This seemingly thermal instability makes sense given the fact that this gene was originally form coral which grows at lower temperatures in the ocean.
Contribution
Contribution by Characterizing the Expression of BBa_K1349002 at Different Temperatures
We used this construction in the characterization of the expression of BBa_K1349002 at different temperatures.
We decided to choose temperature characterization as through our literature review, we found that the source of AmilCP is the coral Acropora millepora. We wondered based on the Violacein’s need for colder conditions for pigment accumulation, if growing these strains at colder conditions allowed for the stronger expression of this pigment. This characterization is new for this version of amilCP (codon optimized for expression in E. coli) as previously temperature different did not impact the original AmilCP from biobrick BBa_K592009. We hoped that with the codon-optimization of this amilCP iGEM Guelph would obtain different results.
Transformants were grown on LB plates containing 50 ng/mL tetracycline, and the plates were placed at 25 °C and 37 °C overnight. The next day, the colonies grown at 25°C were a dark colour, while the colonies grown at 37°C were visibly less pigmented (Figure 10).
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