Team:Pitt/Protocol Design/Intro

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<h2>Protocol Design Intro</h2>
<h2>Protocol Design Intro</h2>
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<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>
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<p>To date, scientists have struggled to find consistent protocols to genetically engineer <i>P. acnes</i>, which is unfortunate given the ubiquitous presence of <i>P. acnes</i> on human skin. Since previous efforts to transform <i>P. acnes</i> have been so unsuccessful, we decided to take as broad of an approach as possible to search for the important variables in a <i>P. acnes</i> transformation protocol. To save on costs and time, a statistical design of experiments (DOX) approach was taken. Using this method, we could test many input parameters <i>concurrently</i> and evaluate their effect on transformation efficiency. Each parameter had two different levels, a high level and a low level. These levels are determined by the experimenter as the range of values that will have a significant effect on the outcome of the experiment. The statistical power of the experiment is not compromised through the use of analysis of variance (ANOVA) tests. One way to visualize this process is if we look at an experiment with three parameters. </p>
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<center><img src = "https://static.igem.org/mediawiki/2014/9/9a/Pitt_dox_intro1.jpg"></center>
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<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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<p>Shown above, each variable has a range which also can interact with the range of values of the other two variables. Each interaction between the three variables produces a certain outcome that is determined by a point in the box. If the whole range of values is tested together for each variable then the whole area of the box or all possible outcomes from the interaction of the three variables can be determined. Assuming there are not any non-linearities (or cats) inside our box, getting a clear outside view of the box will give us a good idea of what the box looks like on the inside (Schrodinger be darned!).</p>
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<center><img src = "https://static.igem.org/mediawiki/2014/e/ee/Pitt_dox_intro2.jpg.png"></center>
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<p><i>P. Acnes</i> is particularly difficult to transform because it has a thick outer cell wall. Unlike the cell pictured above, <i>P. acnes</i> is not as big a fan of taking up plasmid DNA. In addition, the restriction/modification system in <i>P. Acnes</i> is not understood particularly well. Thus, the used parameters emphasized wearing down the cell wall and an attempt to bypass the restriction modification system. We chose 8 factors to manipulate in order to increase the efficacy of transformation in <i>P. Acnes</i>: <i>P. Acnes</i> strain (ATCC 6919 or ATCC 11827), Lysozyme concentration (0.10 mg/ml vs 0.60mg/ml), Glycine concentration (0.50%  or 1.00%), Mass of plasmid DNA (0.70µL or 2.00 µL), Electric field strength (9V or 15V), Culture temperature (24°C or 37°C), Post-transformation temperature (24°C vs 37°C), Presence of restriction enzyme inhibitor TypeOne (present or not present). </p>
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Latest revision as of 19:43, 17 October 2014

Protocol Design Intro

To date, scientists have struggled to find consistent protocols to genetically engineer P. acnes, which is unfortunate given the ubiquitous presence of P. acnes on human skin. Since previous efforts to transform P. acnes have been so unsuccessful, we decided to take as broad of an approach as possible to search for the important variables in a P. acnes transformation protocol. To save on costs and time, a statistical design of experiments (DOX) approach was taken. Using this method, we could test many input parameters concurrently and evaluate their effect on transformation efficiency. Each parameter had two different levels, a high level and a low level. These levels are determined by the experimenter as the range of values that will have a significant effect on the outcome of the experiment. The statistical power of the experiment is not compromised through the use of analysis of variance (ANOVA) tests. One way to visualize this process is if we look at an experiment with three parameters.

Shown above, each variable has a range which also can interact with the range of values of the other two variables. Each interaction between the three variables produces a certain outcome that is determined by a point in the box. If the whole range of values is tested together for each variable then the whole area of the box or all possible outcomes from the interaction of the three variables can be determined. Assuming there are not any non-linearities (or cats) inside our box, getting a clear outside view of the box will give us a good idea of what the box looks like on the inside (Schrodinger be darned!).

P. Acnes is particularly difficult to transform because it has a thick outer cell wall. Unlike the cell pictured above, P. acnes is not as big a fan of taking up plasmid DNA. In addition, the restriction/modification system in P. Acnes is not understood particularly well. Thus, the used parameters emphasized wearing down the cell wall and an attempt to bypass the restriction modification system. We chose 8 factors to manipulate in order to increase the efficacy of transformation in P. Acnes: P. Acnes strain (ATCC 6919 or ATCC 11827), Lysozyme concentration (0.10 mg/ml vs 0.60mg/ml), Glycine concentration (0.50% or 1.00%), Mass of plasmid DNA (0.70µL or 2.00 µL), Electric field strength (9V or 15V), Culture temperature (24°C or 37°C), Post-transformation temperature (24°C vs 37°C), Presence of restriction enzyme inhibitor TypeOne (present or not present).



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