Team:Pitt/Protocol Design/Intro

From 2014.igem.org

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).