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Team:Pitt/Protocol Design/Methods
From 2014.igem.org
DOX Methods
Planning
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.
Electric field strength
The values for electric field strength were chosen based of the Cheong paper.
Culture temperature
We decided to use 24°C because this was the lower end of culture temperature that growth was seen by Cheong. We determined 37°C to be the higher end of the spectrum because this is body temperature.
Post-transformation temperature
We chose the post transformation incubation temperatures for the same reason as the culture temperature.
Presence of restriction enzyme inhibitor (TypeOne)
This particular product was shown to increase the efficiency of transformation by inhibiting type 1 restriction modification systems. Although, there is no confirmed evidence for a type 1 restriction/modification system in P. Acnes
Testing for these 8 parameters required 16 trials, the values of which can be seen below:
Trials
For each trial, the desired P. acnes strain was grown up in approximately 30 mL of A media until attaining an optical density (OD) of at least 0.5. Once at this OD, a specified concentration of glycine was added for a day, then competent cells were made.
The procedure for making competent cells first involved cleaning the bacteria by centrifuging with 10% glycerol buffer. The bacteria was then soaked in 15% glycerol buffer with varying concentration of lysozyme for 2 hours, then was washed again and frozen with dry ice in 50 uL aliquots of 10% glycerol buffer.
Before electroporation, the competent cells were thawed and a specified amount of our desired plasmid (pBRESP36a) was added. At this time, TypeOne restriction enzyme inhibitor was also added for half of the trials. Electroporation then ensued with a set value of electric field strength, after which the bacteria was recovered.
Recovering the bacteria involved letting it grow overnight in 500 uL of A media, then streaking it on A media plates with erythromycin in it. Because of the pBRESP36a, our bacteria would be erythromycin resistant. After a week of growth on the plates, the colonies of bacteria were counted and validated.
Validation
Validating the colonies consisted of ensuring that the bacteria on the plate was erythromycin resistant and that it contained the plasmid, and validating that the bacteria was P. acnes.
To check that the bacterial genome contained our target plasmid, several transformed colonies were restreaked on plates containing erythromycin, then a colony PCR was performed.
To ensure that the bacteria was P. acnes and not some form of contamination, a P. acnes phage was used on a plate with top agar that contained the bacteria.
Timeline
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