Difference between revisions of "Team:British Columbia/Screening"

Imidacloprid (IMI)-transforming bacteria have been isolated from soil (1,2), however the enzymes involved in IMI degradation have not been identified yet. We functionally screened large-insert environmental fosmid libraries obtained from Dr. Hallam for IMI-transforming enzymes, which subsequently could be incorporated into bee gut bacteria (Gilliamella or Snograsella). For the screening we used two approaches – toxicity selective screen and IMI as sole carbon and nitrogen sources.

First screen approach:

We designed this approach to detect IMI-transforming enzymes able to alleviate the toxic concentration of IMI for the E.coli host (as the fosmid libraries constructed in E.coli EPI300 strain host). The screen might identify clones able to modify the toxic compound into less toxic derivatives. First, as there was no information about effect of IMI on E.coli strains, we tested if any concentration of IMI is toxic for the EPI300 strain with concentrations from 0.001 to 10 mM of IMI tested. The cultures were grown in Minimal media with glucose supplemented with different concentrations of IMI. None of the concentrations showed toxicity to thr host as E.coli was able to growth in all experimental conditions, thus we focused on the second screen approach.

Figure 1. Growth of EPPPC1 strain after addition of different concentrations of imidacloprid. The OD₆₀₀ represent the photometric values measured after 24 hours and subtracted from values at 0 hour.

Second screen approach:

IC was used as sole carbon and nitrogen sources for E.coli, therefore the fosmid clones carrying a complete pathway for the IC compound transformation are able to grow. The fosmid clones were tested in pooled format (plate pools of 384-clones and library pools with all clones from each library combined together). The screen didn’t yield clones carrying the target activity.

References:

1. Hu, G., Zhao, Y., Liu, B., Song, F., & You, M. (2013). Isolation of an indigenous imidacloprid-degrading bacterium and imidacloprid bioremediation under simulated in situ and ex situ conditions. Journal of Microbiology and Biotechnology, 23(11), 1617–26.
2. Sabourmoghaddam, N., Zakaria, M. P., & Omar, D. (2015). Evidence for the microbial degradation of imidacloprid in soils of Cameron Highlands. Journal of the Saudi Society of Agricultural Sciences, 14(2), 182–188. http://doi.org/10.1016/j.jssas.2014.03.002
3. Pandey, G., Dorrian, S. J., Russell, R. J., & Oakeshott, J. G. (2009). Biotransformation of the neonicotinoid insecticides imidacloprid and thiamethoxam by Pseudomonas sp. 1G. Biochemical and Biophysical Research Communications, 380(3), 710–4. http://doi.org/10.1016/j.bbrc.2009.01.156