Team:British Columbia/Screening

UBC iGEM 2015

 

Screening

 

Imidacloprid (IMI)-transforming bacteria have been isolated from soil (1,2,3), however the microbial enzymes involved in IMI degradation or transformation have not been identified yet. We functionally screened large-insert environmental fosmid libraries obtained from Dr. Hallam lab for IMI-transforming enzymes, which subsequently could be subcloned and incorporated into bee gut bacteria (Gilliamella or Snograsella). 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 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 0.01 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 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:

As IMI is not toxic to the 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 in the 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 is only caused by the consumption of IMI and not antibiotics or plasmid inducer, the libraries were grown in presence (+) and absence (-)of IMI and all other components were identical between these conditions.

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 screenin. The growth was checked after 48 hours and none of the cultures turned high denseg

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.