Team:British Columbia/Growing
Genetic Tool Development
For the probiotic, the β-proteobacteria, Snodgrassella alvi, and the γ-proteobacteria, Gilliamella apicola, were chosen since it is distinctly endogenous to the midgut of the European honey bee, Apis mellifera (1). Since these microaerophiles are native and unique to the honey bee gut, introducing imidacloprid and 6-CNA degradation genes into these candidate bacteria would minimize the chance of resistance genes spreading to other insects. Due to the limited amount of existing literature on G. apicola and S. alvi, the project focused on discovering methods to make these bacteria genetically tractable. This included culturing the bacteria on different growth mediums, testing methods to induce competence, and transformation techniques with a variety of plasmids.
Culturing:
Due to the novel nature of using G. apicola and S. alvi for the project (as opposed to E. coli), the 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 blood) were generously provided by Waldan K. Kwong from Yale University. Initially, a variety of media was tried to culture S. alvi and G. apicola: LB plates, TSA plates and blood agar plates. Following the methods from Kwong et al. (1), streaked agar plates were incubated in an anaerobic jar equilibrated with 5% CO2 balanced with N2. G.apicola grew best on TSA plates and S. alvi grew best on blood agar plates incubated at 37°C. G.apicola plates showed growth after 48 hrs, whereas S. alvi plates showed growth after four days. Further tests to induce competence for transformation were performed with G. apicola due to the faster growth rate and larger colony size.
The various liquid media tested included brain heart infusion broth, Mueller Hinton broth, LB, Tryptic Soy Broth (TSB), and Super Optimal Broth with Catabolite repression (SOC). Media was prepared in a sealed Hungate tube and flushed with 5% CO2 balanced with N2 gas to remove O2 that was dissolved in the media. Using a syringe, media was inoculated with G. apicola or S. alvi and incubated at 37°C for 48 hrs. S. alvi was not culturable in any of the liquid media tested. G. apicola did grow under anaerobic conditions in TSB requiring 48 hrs to form visible growth.
Growth Curve
The growth curve of G. apicola was monitored on a plate reader that measured the OD value at 600nm over 36 hours. G. apicola was inoculated into a TSB culture that was previously flushed with 5% CO2 balanced with N2. Additionally, 5% CO2 balanced with N2 was blown onto the plate whilst sealing to ensure the lowest possible amount of oxygen was present in the plate.
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, various protocols known to work on other gram-negative gammaproteobacteria, as well as a protocol for microaerophilic bacteria were attempted.
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Transforming
Acknowledgements
References
UBC iGEM 2015