Try, Try, and Try Again

When our team initially cloned the Ben clusters from Acinetobacter baylyi and Rhodococcus into the E. coli strain, the transformation was unable to use benzoate as a carbon source. At first, we tried different concentrations of benzoate in the media but there was still no growth. We went back to our original cloning process, and we noticed that the ribosome binding site was too far away from the start codon. Once we had noticed this, we redesigned our primers for PCR amplification and cloning so that this could be corrected. We have successfully cloned 6 genes from A. baylyi and Rhodococcus Op-like and one full gene cluster from A. baylyi. We had similar troubles when attempting to grow another Rhodococcus strain in minimal media with benzene as a carbon source. In this case however, we were able to follow a different method and use a new media to see growth on benzene as a carbon source.