Team:DeNovocastrians/Results
benE
The benE transporter gene was extracted and amplified from three bacterial sources. The benE gene from both Rhodococcus strain 9 and Acinetobacter baylyi ADP1 was obtained from genomic DNA. The benE from Escherichia fergusonii was ordered and synthesised from the IDT laboratory. Gene amplification was successfully completed through the use of PCR.
The amplified benE PCR products from E. Fergusonii and Rhodococcus strain 9 were successfully cloned into the pTTQ18 plasmid.
The pTTQ18 vector with benE inserts were transformed into the E.Coli strain BW25113 with the native benzene transporter knocked out (ΔydcO). The transformants were used in growth curve assays to assess the import ability of the benE transporter and can be seen in the figure below.
The transformed E. coli with the benE gene were grown in the presence of 15mM benzoate. The benE transformants (with parts BBa_K3694027 and BBa_K3694028) performed better than the BW25113 ΔydcO control, indicating the transporter was functioning and importing benzoate from solution.
High powered liquid chromatography (HPLC) was performed on the LB + benzoate growth curve samples used in the growth curve experiment. The cells were removed from the solution and analysis was undertaken on the concentration of benzoate left in solution. The benE transformants had less benzoate left in solution compared to the BW25113 ΔydcO control. This confirms the BBa_K3694028 and BBa_K3694028 benE biobrick parts were successfully importing benzoate from solution into the cell.
HPLC was also used to determine the presence of catechol of the growth curve solutions as it is the end-point of the benzene degradation pathway. There was confirmed to catechol in the transformant with the BBa_K3694029 part. This means that the E. coli were importing and degrading benzoate, further confirmation of our successful cloning and expression of our benE biobrick parts.
benABCD
The benABCD genes were extracted and amplified from Rhodococcus strain 9 and Acinetobacter baylyi ADP1 through PCR. The amplified PCR products were digested and ligated into the pTTQ18 plasmid. A digestion check suggested the pTTQ18::benABCD(strain 9) vector had successfully incorporated the gene insert. The pTTQ18::benABCD(strain 9) vector was transformed into the E.coli strain BW25113. However, the growth curve analysis undertaken on the transformed BW25113 cultures found no degradation of benzoate. The pTTQ18::benABCD(strain 9) may have been incorrectly incorporated or mutated through the cloning process. Sequencing data would be required to ensure the benABCD gene is correctly cloned into the pTTQ18 plasmid.
Biosensor
Our initial attempts to join DNA fragments (fluorescent protein genes and benzene/catechol regulatory sequences) together for the construction of our biosensor were unsuccessful. However, by ordering new primers, increasing annealing temperature during PCR and using an optimisation protocol set by Hilgarth and Lanigan (2020), we joined some fragments together. We were able to join catM binding region to mCherry fluorescent gene, the catM gene to the catM promoter, and the benM gene to thebenM binding region. After this successful joining, we aimed to transform these constructed parts into organism for testing. Unfortunately, we were unable to produce viable transformed colonies and were therefore unable to perform any colony pcr to confirm the full construction of our biosensor.
Future targets for benABCDE transformation
We identified two potential insertion targets in: the marine organism Synechocytis cyanobacteria, and, the common terrestrial organism Bacillus subtilis. Neither species were able to grow in/degrade benzoate in their native states. In the image below: A) B. subtilis grew in control media (containing glucose) but was unable to grow in 10mM/100mM benzoate; B) Synechocystis cyanobacteria grew in control media (BG-11) but was unable to grow in 10mM/100mM benzoate. Therefore, they are potential targets for the insertion of the benABCDE cluster in future projects.
Hilgarth, R. S. and Lanigan, T. M. (2020). Optimization of overlap extension PCR for efficient transgene construction. MethodsX 7:100759.
