Team:Pitt/HSP60 Promoter/Intro

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Latest revision as of 10:42, 17 October 2014

HSP60 Promoter Intro

Essential to using Propionibacterium acnes as a new chassis for synthetic biology applications was to first identify genetic regulatory elements that could lead to efficient transgene expression in P. acnes. P. acnes is very distantly related to E. coli, belonging to a different phylum. Thus, we hypothesized that E. coli promoters would not function in P. acnes. Additionally, while some P. acnes promoters have predicted in the recently sequenced genome, none have been cloned to date.(Ref 1) Therefore we sought to find promoter elements from the closely-related and well-characterized mycobacterium M. bovis. Specifically, we cloned the Hsp60 promoter from M. bovis, a promoter known to be highly active in mycobacteria and to function in other propionibacteria.(Ref 2) As we didn’t know what RBS’s would be optimal for P. acnes we generated two versions of the promoter - one which contained only the promoter sequence and no RBS (Part: BBa_K1548000), and another which contained the promoter and the strong RBS from bacteriophage TM4 (Part: BBa_K1548001), known to lead to high levels of translation in mycobacteria.

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 <h2>HSP60 Promoter Intro</h2>
-<p>To reach high levels of protein expression, the Pitt iGEM Team is isolating a high-copy promoter, HSP60, for our BioBrick parts. The HSP60 protein is a ubiquitous chaperone, helping proteins fold correctly, and is necessary for cell survival. Therefore, the HSP60 promoter needs to produce a large amount of the HSP60 protein to prevent other critical proteins from misfolding. Consequently, the HSP60 promoter is an excellent choice for maximum expression BioBrick parts. The Pitt iGEM is using the HSP60 promoter in devices to <a href="https://2014.igem.org/Team:Pitt/Skin_Probiotic/Dehydrogenase">balance skin oil</a> and to <a href="https://2014.igem.org/Team:Pitt/Skin_Probiotic/Melanin">produce melanin</a>.</p>
+<p>Essential to using <i>Propionibacterium acnes</i> as a new chassis for synthetic biology applications was to first identify genetic regulatory elements that could lead to efficient transgene expression in <i>P. acnes. P. acnes</i> is very distantly related to <i>E. coli</i>, belonging to a different phylum. Thus, we hypothesized that <i>E. coli</i> promoters would not function in <i>P. acnes</i>. Additionally, while some P. acnes promoters have predicted in the recently sequenced genome, none have been cloned to date.(Ref 1) Therefore we sought to find promoter elements from the closely-related and well-characterized mycobacterium M. bovis. Specifically, we cloned the Hsp60 promoter from <i>M. bovis</i>, a promoter known to be highly active in mycobacteria and to function in other propionibacteria.(Ref 2) As we didn’t know what RBS’s would be optimal for <i>P. acnes</i> we generated two versions of the promoter - one which contained only the promoter sequence and no RBS (Part: BBa_K1548000), and another which contained the promoter and the strong RBS from bacteriophage TM4 (Part: BBa_K1548001), known to lead to high levels of translation in mycobacteria. </p>
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