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Team:Pitt/Protocol Design/Methods
From 2014.igem.org
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<h2 id = "methods">Methods</h2> | <h2 id = "methods">Methods</h2> | ||
| - | < | + | <p>We chose to evaluate 8 parameters, designating high and low values for each. These values were chosen carefully from reading through current literature on <i>P. acnes</i> and other electroporation protocols. The 8 parameters were:</p> |
| - | <p> | + | <p><u>P. Acnes strain</u></p> |
| - | < | + | |
| + | <p>We chose to use two different types of <i>P. Acnes</i> 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 <i>P. Acnes</i> was used because there is CRISPR/Cas system in this class. This system protects <i>p. Acnes</i> against plasmid and foreign DNA.</p> | ||
| + | |||
| + | <p><u>Lysozyme concentration</u></p> | ||
| + | |||
| + | <p>Since, <i>P. Acnes</i> 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 <i>P. Acnes</i>.</p> | ||
| + | |||
| + | |||
| + | <p><u>Glycine concentration</u></p> | ||
| + | <p>Similar to the lysozyme, glycine was used as a cell wall weakening agent. The concentrations were determined from a paper which transformed other <i>P. Acnes</i> bacteria. (Fan)</p> | ||
| + | |||
| + | |||
| + | <p><u>Mass of plasmid DNA</u></p> | ||
| + | |||
| + | <p>The high and low values for this parameter were based of values suggested by a protocol written by UCLA and a protocol by Cheong.</p> | ||
| + | <center> | ||
| + | <img src = "https://static.igem.org/mediawiki/2014/3/30/Pitt_dox_methods1.jpg"></center> | ||
<hr> | <hr> | ||
Revision as of 11:39, 17 October 2014
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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