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.
G. apicola and S. alvi were streaked on TSA, LB, and blood agar (5% sheep) plates and stored and grown at 37°C in an anaerobic jar flushed with 5% CO2 balanced with N2. Growth was tested on a variety of media types in order to determine the best growing conditions. Based on the visual indication of colony size, number, and growing time, G. apicola grew the best on TSA plates, while S. alvi grew best on blood agar (5% sheep blood). G. apicola and S. alvi colonies gave distinct colonies after 48 and 96 hours, respectively, in the microaerophilic chamber.
Figure 1: S. alvi on a 5% sheep blood agar plate. Figure 2: G. apicola on a TSA plate. As no liquid growth medium for S. alvi or G. apicola have been reported in literature, a variety of liquid media growth conditions were tested (see Table 1). All liquid cultures were incubated at 37°C for 72 hours, or until a turbidity was visibly detected. G. apicola grew in TSB with an anaerobic environment. Whilst growing in liquid media, it was noted that G. apicola aggregated into dense snowflake-like colonies (Figure 3). S. alvi was not successfully cultivated in any liquid media tested. Colony PCR and plating on oxytetracycline plates under microaerophilic conditions were used to confirm growth was G. apicola or S. alvi.
Figure 1: S. alvi on a 5% sheep blood agar plate. Figure 2: G. apicola on a TSA plate. | Liquid Media | Method | S. alvi | G. apicola |
|---|---|---|---|
| TSB | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| LB | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| SOC | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| MH Broth | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| Brain Heart Infusion | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| TSB | Flushed with 5% CO2 Balanced with N2 while hot | No Growth | Successful Growth |
Table 1: Types of liquid media tested for growth of S. alvi and G. apicola.
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.
The growth curve of G. apicola in a TSB culture was monitored on a plate reader that took OD values at 600nm. As displayed in figure 3 G. apicola’s lag phase lasts 15 hours. Moreover, due to it’s slow growth, it takes approximately 24 hours to reach a stationary-like growth phase.
Figure 1: S. alvi on a 5% sheep blood agar 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. View our protocols here, under Genetic Tool Development.
Two protocols were attempted for creating electrocompetent cells: one designed for Campylobacter jejuni (similar to G. apicola because it is a microaerophilic bacteria) and one designed for Salmonella (similar to G. apicola because it is also a γ-proteobacteria).
Following the protocol from Methods in Microbiology: Bacterial Pathogenesis for Campylobacter jejuni by Williams, P., Ketley, J., & Salmond, G. (2), G. apicola was grown on TSA for 48 hours at 37°C, after which the biomass was removed. Cells were washed with ice cold wash buffer of sucrose and glycerol. Competent cells were then stored at -80°C, or transformed immediately by electroporation.
For the second method, the protocol from Methods in Microbiology, Vol. 47: Electroporation Protocols for Microorganisms (Salmonella) by Nickoloff, J. A. (3), was used to induce competence in G. apicola after 48 hrs of growth at 37°C. Biomass was harvested and washed with HEPES buffer and 10% glycerol. Competent cells were either stored at -80°C or transformed immediately by electroporation.
One protocol for creating chemically competent cells was attempted: designed for E. coli (similar to G. apicola because it is also a γ-proteobacteria). G. apicola was grown on TSA for 48 hours at 37C, after which the biomass was removed. Cells were then washed with a CaCl2 buffer. Competent cells were stored at -80C, or transformed immediately by heat shock.
Transformation
After creating the competent cells, we attempted a variety of transformation protocols. View our protocols here, under Genetic Tool Development.
Acknowledgements
We would like to thank the following people greatly for their assistance, suggestions, and providing the plasmids/materials for us to experiment with.
Walden Kwong for providing the strains of G. apicola and S. alvi.
Dr. Julian Davies for providing the RP1 plasmid.
Dr. John Smit and Dr. John Nomellini for providing the E.coli S17, and SM-10 strains. As well for providing the plasmids PBBR3, PBBR4, PKT210, and PRK293.
Dr. Rachel Fernandez for providing the PBBRMCS1-2 plasmid.
Dr. J. Thomas Beatty for providing the PIND4 plasmid.
Dr. Bob Hancock and Dr. Mangeet Bains for providing PBBR1MCS-3, PBBR1MCS-5, and PBSPIISK(-).
Dr. Michael Murphy and everyone in the Murphy Lab for being amazing hosts.
References
- Kwong, W., Engel, P., Koch, H., and Moran, N. (2014). Genomics and host specialization of honey bee and bumble bee gut symbionts. Proceedings of the National Academy of Sciences, 111, 11509-11514.
- Williams, P., Ketley, J., & Salmond, G. (Eds.). (1998). Bacterial Pathogenesis. London, UK: Academic Press.
- Nickoloff, J. A. (Ed.). (1995). Electroporation Protocol for Microorganisms. Totowa, NJ: Humana Press Inc.
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- Rodriguez, R. L, & Denhardt, D. T. (1988). Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Stoneham, MA: Butterworth Publishers.
- Plasmid map of pIND4 for Rhodobacter sphaeroides. (2005). Retrieved August 5, 2015.
- Schweizer, H. P. (2001). Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads. Curr. Opin. Biotechnol. 12:439–445.
- Kovach, M.E., Elzer, P.H., Steven Hill, D., Robertson, G.T., Farris, M.A., Roop, R.M., and Peterson, K.M. (1995). Four New Derivatives of the Broad-Host-Range Cloning Vector pBBR1MCS, Carrying Different Antibiotic-Resistance Cassettes. Gene, 166(1). 175-176.
UBC iGEM 2015