Team:Pitt/Protocol Design/Intro

Protocol Design Intro

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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.

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.

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