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

Imidacloprid (IMI)-transforming bacteria have been isolated from soil (1,2,3), however the microbial pathways involved in IMI degradation or transformation have not yet been characterized. We functionally screened large-insert environmental fosmid libraries obtained from the Hallam lab at UBC for IMI-transforming enzymes, which subsequently could be subcloned and incorporated into bee gut bacteria (Gilliamella or Snodgrasella). For the functional 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 on 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 0.01 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 the host, as E.coli was able to grow in all experimental conditions. As a result, we focused on the second screen approach.

Figure 1. Growth of EPCC1 strain after addition of different concentrations of imidacloprid. The OD₆₀₀ represent the photometric values measured after 24 hours and subtracted from photometric values at 0 hour.

Second screen approach:

As IMI is not toxic to E.coli and the compound contains nitrogen and carbon, it was used as sole carbon and nitrogen sources in the screening of fosmid libraries derived from soil environments. Presumably, only the fosmid clones carrying a complete pathway for the IMI compound transformation into TCA cycle intermediates would be able to grow under these conditions. 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 pooled fosmid clones were grown in minimal media without glucose for IMI as carbon source screening approach (Figure 2), or without ammonium chloride for nitrogen source screening approach (Figure 3). To be sure that growth was only due to the consumption of IMI rather than antibiotics or plasmid copy inducer, the libraries were grown in presence (+) and absence (-) of IMI and all other components were identical between these conditions. The screen did not identify any clones showing the IMI-degrading phenotype, likely due to the fact that either the pathway was not present in screened libraries or that the E.coli host was not able to express it.

Figure 2. Growth results for IMI as sole carbon source screening. The growth was checked after 48 hours and none of the cultures turned high dense.

Figure 3. Growth results for IMI as sole nitrogen source screening. The growth was checked after 48 hours and none of the cultures turned high dense.

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.
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.