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

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<p>IC was used as sole carbon and nitrogen sources for <i>E.coli</i>, 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.</p>
 
<p>IC was used as sole carbon and nitrogen sources for <i>E.coli</i>, 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.</p>
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<li>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.</li>
 
<li>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.</li>
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<li>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. <a href="http://doi.org/10.1016/j.bbrc.2009.01.156">http://doi.org/10.1016/j.bbrc.2009.01.156</a></li>
 
<li>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. <a href="http://doi.org/10.1016/j.bbrc.2009.01.156">http://doi.org/10.1016/j.bbrc.2009.01.156</a></li>
 
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Revision as of 23:46, 30 August 2015

UBC iGEM 2015

 

Screening

 

Imidacloprid (IC)-transforming bacteria have been isolated from soil (1,2), however the enzymes involved in IC degradation have not been identified yet. We screened large-insert environmental fosmid libraries obtained from Dr. Hallam for IC-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 IC as sole carbon and nitrogen sources.

First screen approach:

We designed this approach to detect IC-transforming enzymes able to alleviate the toxic concentration of IC 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. We tested what concentrations of IC are toxic for the E.coli strain with concentrations from 0.01% to 2% (w/v) tested. None of these showed toxicity to E.coli strain thus we focused on the second screen approach.

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