Team:Pitt/HSP60 Promoter/Results

From 2014.igem.org

Results and Conclusions for HSP60

We successfully cloned the Hsp60 promoter and RBS from M. Bovis and bacteriophage TM4, respectfully, which we expect to function in P. acnes. While waiting for the protocol to be developed for transformation of P. acnes with plasmid DNA, we tested whether the promoter would be functional in E. coli, by assaying expression of the mRFP1 protein. The hsp60 promoter was cloned upstream of the B0034 RBS and mRFP1 gene comprising the part Part: BBa_K1548002 as pictured in Figure 2.

Figure 2. The hsp60 promoter mRFP1 protein generator BBa_K1548002

We found that the hsp60 promoter was not functional in E. coli as it did not lead to mRFP1 production.

Figure 3. The hsp60 promoter does not produce mRFP1 in E. coli.

We then ran an E. coli promoter prediction software BPROM on (http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) which validated our result that the promoter was non-functional in E. coli. We are confident that the promoter will be highly active in P. acnes based on experiments performed in closely related bacteria in Ref. 1. Once we optimize the transformation of P. acnes we can test the efficacy of this promoter in P. acnes.

References

1. Brüggemann H, Henne A, Hoster F, Liesegang H, Wiezer A, Strittmatter A, Hujer S, Dürre P, Gottschalk G. The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Science. 2004 Jul 30;305(5684):671-3.

2. Oldfield LM, Hatfull GF. Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression. J Bacteriol. 2014 Oct 15;196(20):3589-97. doi: 10.1128/JB.01801-14. Epub 2014 Aug 4.