Team:Pitt/Protocol Design/Methods

Methods

We chose to evaluate 8 parameters, designating high and low values for each. These values were chosen carefully from reading through current literature on P. acnes and other electroporation protocols. The 8 parameters were:

P. Acnes strain

We chose to use two different types of P. Acnes strains to determine if this made a significant difference in the transformation efficiency. We did however restrict the strain to being dam- strains because unmethylated DNA was shown to have greater transformation efficiency (Cheong). In addition, Type I P. Acnes was used because there is CRISPR/Cas system in this class. This system protects p. Acnes against plasmid and foreign DNA.

Lysozyme concentration

Since, P. Acnes has a particularly thick cell wall, lysozyme was used as a cell wall weakening agent. Lysozyme is a common cell wall weakening agent and was readily available. The concentrations were determined from previous experiments in lab with P. Acnes.

Glycine concentration

Similar to the lysozyme, glycine was used as a cell wall weakening agent. The concentrations were determined from a paper which transformed other P. Acnes bacteria. (Fan)

Mass of plasmid DNA

The high and low values for this parameter were based of values suggested by a protocol written by UCLA and a protocol by Cheong.

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