Difference between revisions of "Team:British Columbia/Growing"
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| + | <h4>Inducing Competence in <i>G.apicola</i> and <i>S.alvi</i></h4> | ||
| + | <p>After identifying the optimal way to culture <i>G. apicola</i>, we moved on to attempting various ways of inducing competence in the bacteria. Due to no existing literature on methods of inserting a plasmid into <i>G. apicola</i>, we attempted various protocols known to work on other gram-negative gammaproteobacteria, as well as a protocol for microaerophilic bacteria. | ||
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| + | <h4>Method One</h4> | ||
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Revision as of 05:33, 1 September 2015
Growing
For our probiotic, we chose the betaproteobacteria, Snodgrassella alvi, and the gammaproteobacteria, Gilliamella apicola, both of which are specific to Apis mellifera. By implementing our system in these microaerophiles which are native and unique to the honey bee gut, we are inhibiting other insects from acquiring our engineered, imidacloprid resistant strains. However, due to the small amount of existing literature on G. apicola and S. alvi, our project revolved around discovering methods of culturing the bacteria, inducing competence, and transforming them with a compatible plasmid.
Culturing:
Due to the novel nature of using G. apicola and S. alvi for the project (as opposed to E. coli), our first step was to determine the optimal method of culturing either bacteria.
The bacterial strains, plated on blood agar (tryptic soy agar supplemented with 5% sheep’s blood) were generously provided by Waldan K. Kwong from Yale University. Initially, we tried a variety of methods of culturing both S. alvi and G. apicola: LB plates, TSA plates, blood agar plates (5% sheep blood). Following the methods from Kwong et al., we incubated the inoculated agar plates in a microaerophilic chamber flushed with 5% CO2 balanced with N2. After numerous trials of growing the bacteria on different media, we determined G.apicola grew best on TSA plates and S.alvi grew best on blood agar plates. G.apicola plates showed significant growth after two days, whereas S.alvi plates showed significant growth after four days. Due to G. apicola’s faster growth rate and its larger colony diameter, we decided to proceed with the transformations using G. apicola.
Inducing Competence in G.apicola and S.alvi
After identifying the optimal way to culture G. apicola, we moved on to attempting various ways of inducing competence in the bacteria. Due to no existing literature on methods of inserting a plasmid into G. apicola, we attempted various protocols known to work on other gram-negative gammaproteobacteria, as well as a protocol for microaerophilic bacteria.
UBC iGEM 2015