We have determined the toxicity of linuron and 3,4-Dichloroaniline (3,4-DCA) toward B. diazoefficiens using growth curve Minimum Inhibitory Concentration (MIC) experiments, with IC50 values of 24.01 and 20.82 µM respectively. These values confirm that linuon is indeed toxic to B. diazoefficiens at agriculturally-relevant concentrations (Fernandez-Pascual, 1988). This information demonstrates the need for a more tolerant engineered organism. The collected toxicity data also provides benchmarks against which to compare the performance of any future engineered strains.
Through our experiments, we have shown that E. coli DH5α is not inhibited by linuron concentrations up to linuron’s solubility limit, 290 µM. We have also shown that E. coli DH5α is not inhibited by 3,4-DCA concentrations up to 226 µM, with some inhibition occurring above this concentration. We have demonstrated that LibA and NAT1 genes in the pRJPaph-GFP vector do not impose a metabolic burden on DH5α compared to the empty vector alone. However, LibA and NAT1 do not lend increased tolerance to LibA or NAT1 versus the empty vector in E. coli. This does not necessarily mean that our system doesn't work as genes were optimized for expression in B. diazoefficiens, not E. coli.