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Team:Cornell/project/wetlab/assembly/naphthalene
From 2012.igem.org
Naphthalene Reporter
Background and Previous Naphthalene Sensors
The NAH7 plasmid common among Pseudomonas spp. contains two operons. The nah operon encodes enzymes that degrade naphthalene to salicylate, while the sal operon encodes enzymes that degrade salicylate into common cell metabolites. Naphthalene degradation by the nah and sal gene products is regulated at the transcriptional level by NahR [1,2]. Schell and Wender, 1986, showed that NahR, a 36-kDa activator protein, is constitutively expressed and tightly bound upstream of the nah and sal operon in the presence and absence of salicylate. Yet, when salicylate is present, expression of nah and sal is significantly increased. The current model of NahR-mediated activation hypothesizes that in the presence of its inducer, salicylate, NahR undergoes a conformational change, increasing the affinity of RNA polymerase to the promoter region, and upregulating the expression of nah and sal several fold [1,2]. Because the nah gene products degrade naphthalene to salicylate, this in turn creates a positive feedback loop promoting expression of the nah and sal operons [3].Although no previous iGEM team has worked on a naphthalene sensor, Valdman, Battaglini, Leite, and Valdman, 2004, previously used a lux-nahG fusion strain to create a sensitive and accurate bioluminescence-based detection system.
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.
We inserted our arsenic-sensing BioBrick into the pBBRBB cloning vector (Vick et al., 2011) due to its past success in complementation studies [7]. Upon ligation, we transformed competent DH5a E. coli and attempted to transform JG700, our mtrB knockout strain. Because we had no successes electroporating JG700 with our construct, we decided to append a mobility gene and use conjugation to transfer our construct. We first transformed WM3064 with our mobility-enabled arsenic sensing construct. Finally, we conjugated WM3064 with JG700 to create our arsenic-sensing Shewanella strain.
References
1. Schell, M.A., and Wender, P.E. (1986). Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. Journal of Bacteriology 166(1): 9-142. Schell, M. A., & Poser, E. F. (1989). mutational analysis of NahR protein binding to Demonstration , Characterization , and Mutational Analysis of NahR Protein Binding to nah and sal Promoters, 171(2).
3. Park, W., Padmanabhan, P., Padmanabhan, S., Zylstra, G. J., & Madsen, E. L. (2002). nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA. Microbiology 148(8): 2319-2329
4. Valdman, E., Battaglini, F., Leite, S. G. F., & Valdman, B. (2004). Naphthalene detection by a bioluminescence sensor applied to wastewater samples. Sensors and Actuators B: Chemical, 103(1-2), 7-12. doi:10.1016/j.snb.2004.01.017
5. King, J. M. H., DiGrazia, P. M., Applegate, B., Burlage, R., Sanseverino, J., & al, e. (1990). Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science, 249(4970), 778-778.
