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Achievements

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We characterised LL-37 antimicrobial peptide from Team Trieste 2008. They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation.

The principal aim of our project was to collaborate as much as possible to the iGEM mission to develop science freely. The parts we designed followed this principle, by being useful in very aspects, apart of our own project. We developed a Multiple Antibiotic Resistance protein, which acts as a transcriptional activator in E. coli, it allows the expression of the acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to organic solvents, dyes, detergents, antibiotics such as chloramphenicol, tetracycline, novobiocin, erythromycin, fusidic acid and cloxacillin, as well as to cationic antimicrobial peptides, such as LL-37, HNP-2 and HBD-1. This part can be used in several ways in different iGEM projects. We also sent to the parts registry the LL-37 peptide, with adequate codon usage, an improvement on part BBa_K875000, that allows LL-37 expression.

As part of our collection, we also designed the device BBa_K1230009, which is the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction, with the Elowitz repressilator. This part will allow an easy way to produce proteins by Arabinose induction, improving the way characterization is made on iGEM.