Team:British Columbia/Growing

UBC iGEM 2015

 

Genetic Tool Development

 

The β-proteobacteria, Snodgrassella alvi, and the γ-proteobacteria, Gilliamella apicola, were chosen as candidates for our probeeotic due to their endogenous nature in relation to the midgut of the European honey bee, Apis mellifera (1). Native and unique to the honey bee gut, the introduction of 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 media, testing methods of competence induction, and transformation techniques with a variety of plasmids.

Culturing

Due to the novelty of using G. apicola and S. alvi for the project (vs. E. coli), the first step was to identify the optimal method of culturing either bacteria.

Growth Curve

The growth of G. apicola was monitored on a plate reader that measured the OD value at 600nm over 36 hours, and plotted to a curve at fixed time points. For this, 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 presence of a minimal amount of oxygen in the plate.

Inducing Competence in G.apicola and S.alvi

After identifying the optimal method to culture G. apicola, we moved on to attempting various ways of inducing competence in the bacteria. Due to the lack of existing literature on methods of inserting a plasmid into G. apicola, we tried various protocols known to work on other gram-negative gammaproteobacteria, and a protocol for microaerophilic bacteria were attempted. View our protocols here, under Genetic Tool Development.

Transformation

After creating the competent cells, a variety of transformation protocols were attempted. 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

  1. 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.
  2. Williams, P., Ketley, J., & Salmond, G. (Eds.). (1998). Bacterial Pathogenesis. London, UK: Academic Press.
  3. Nickoloff, J. A. (Ed.). (1995). Electroporation Protocol for Microorganisms. Totowa, NJ: Humana Press Inc.
  4. Van der Geize, R. et al. (2002). Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Molecular Microbiology, 45(4). doi:0.1046/j.1365-2958.2002.03069
  5. Koch, H. et al. (2013). Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Molecular Ecology, 22(7). doi: 10.1111/mec.12209
  6. Cho, J. et al. (2003). The Effects of Altering Autoinducer-2 Concentration on Transfer Efficiencies of the F and RPI plasmids to the Quorum Sensing Recicpient Escherichia coli Strain AB1157. Journal of Experimental Microbiology and Immunology (JEMI), 3, pp. 8-14.
  7. Chan, V. et al. (2002). The Effect of Increasing Plasmid Size on Transformation Efficiency in Escherichia coli. Journal of Experimental Microbiology and Immunology (JEMI), 2, pp. 207-223.
  8. Rodriguez, R. L, & Denhardt, D. T. (1988). Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Stoneham, MA: Butterworth Publishers.
  9. Plasmid map of pIND4 for Rhodobacter sphaeroides. (2005). Retrieved August 5, 2015.
  10. Schweizer, H. P. (2001). Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads. Curr. Opin. Biotechnol. 12:439–445.
  11. 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.