Team:Pitt/Skin Probiotic/Cathelicidin/Intro

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Revision as of 16:58, 9 October 2014

Cathelicidin

The natural tendency for P. acnes to reside in the skin makes P. acnes an ideal candidate for a skin probiotic. Using the transformation protocol optimized by our team, we can insert novel genes for beneficent functions into P. acnes. One such function is the secretion of an anti-microbial peptide, which would reduce inflammation by inhibiting the growth of bacteria in the area.

Cathelicidin is a natural antibiotic produced by the immune cells of many mammals, including humans. The antimicrobial peptide gene will be constructed into the plasmid along with an inducible blue-light promoter. If successful, the bacteria containing the plasmid will produce the cathelicidin when exposed to blue light and in turn suppress the P. acnes population when it becomes elevated.

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-<h2>Protocol Design Intro</h2>
+<h2>Cathelicidin</h2>
-<p>In order to insert genes into P. acnes, we need to establish a proper transformation protocol. However, P. acnes are notoriously hard bacteria to transform because of the strength of the cell wall, and because of the wide range of restriction enzymes present in the cytoplasm. Our general approach is to weaken the cell wall of P. acnes during transformation, increasing the amount of DNA that will enter the cell, and to inhibit the restriction enzymes of P. acnes, increasing the amount of DNA that will stay in the cell. In addition, the plasmid used for our experiments, pBRESP36a, originates from a unique bacterium closely related to P. acnes, whereas most of the iGEM Bio-Bricks originate from E. coli.</p>
+<p>The natural tendency for P. acnes to reside in the skin makes P. acnes an ideal candidate for a skin probiotic. Using the transformation protocol optimized by our team, we can insert novel genes for beneficent functions into P. acnes. One such function is the secretion of an anti-microbial peptide, which would reduce inflammation by inhibiting the growth of bacteria in the area.</p>
+<p>Cathelicidin is a natural antibiotic produced by the immune cells of many mammals, including humans. The antimicrobial peptide gene will be constructed into the plasmid along with an inducible blue-light promoter. If successful, the bacteria containing the plasmid will produce the cathelicidin when exposed to blue light and in turn suppress the P. acnes population when it becomes elevated.</p>
-<p>We are using a 2-level, partial factor, statistical design of experiments (DOX) to optimize the transformation efficiency of P. acnes through electroporation. Through DOX, we can maximize the number of variables tested, while minimizing the number of trials run. For this project, we decided to evaluate 8 different parameters: The strain of P. acnes, temperature in which P. acnes is grown, amount of glycine added, amount of lysozyme, amount of the plasmid DNA (pBRESP36A), presence of restriction enzyme inhibitor (TypeOne), strength of electric field, and the incubation temperature for recovery. Optimizing the transformation protocol of P. acnes greatly increases the ease with which researchers can study P. acnes by transforming P. acnes to perform other functions. Furthermore, these novel functions can also be used to turn P. acnes into a skin probiotic, by secreting compounds beneficent for the skin.</p>
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 <a href = "https://2014.igem.org/Team:Pitt/HSP60_Promoter/Results">