Team:Cornell/testing/project/wetlab/3/2

Design and Construction of our Naphthalene Sensors

As with the design of our initial arsenic reporter, we employed two existing BioBricks in the construction of our naphthalene reporter system. BBa_K098994 – which encodes mtrB – will be driven by a naphthalene sensitive system, BBa_K2280004 (PKU Beijing, 2009). This system includes the transcriptional activator nahR, as well as the inducible promoter region, Psal. Construction of our salicylate-sensing construct was expedited by exploiting the modularity of our arsenic-sensing construct. We appended our salicylate-sensitive system upstream of mtrB by a single restriction enzyme digestion and ligation step, into the final backbone, and have successfully conjugated our salicylate sensor into JG700.

Because NahR is indirectly activated by naphthalene through a degradation product, salicylate, our reporter strains must also express the proteins needed for naphthalene catabolism (Schell & Wender, 1986). We therefore, isolated the ≈ 9.5 kb nah operon from the NAH7 plasmid of Pseudomonas putida G7. Due to the sheer size of the operon, we hypothesized that cloning nah into the same plasmid as our salicylate reporter system, pBBRBB, would be simultaneously difficult to accomplish and stressful to our engineered Shewanella strains. The nah operon also contains internal cut sites for NotI and PstI, making it BioBrick incompatible. We decided to try Gibson assembly as a means of assembling and mutagenizing the operon to remove the internal cut-sites. Although we initially thought our assembly was successful, sequencing results showed the contrary.

We appended a constitutive promoter upstream of the first coding sequence of the operon and an origin of transfer before ligation into pSB3C5. Any naphthalene entering our engineered cells will be degraded to salicylate; salicylate will then activate nahR and upregulate expression of mtrB. Because the nah gene products degrade naphthalene to salicylate, this BioBrick also has the potential for future work in optimizing bacterial degradation of PAHs. Due to problems of slow growth with our transformed WM3064 strains, we are still in the midst of confirming the successful conjugation of our nah construct into JG700. Furthermore, we are performing site-directed mutagenesis to make our construct BioBrick compatible.

We attempted to conjugate the nah operon into MR-1 by making a new plasmid with the nah operon in a backbone containing a unique origin of transfer. The origin of transfer was unique in that it differs from the mob origin of transfer in the salicylate reporter plasmid - plasmids with the same origin of transfer cannot be simultaneously maintained in one cell. However, we ran into difficulties conjugating due to slow growth, perhaps derived from the stress of expressing such a large operon within a cell.