We collaborated with the Pitt iGEM team (https://2017.igem.org/Team:Pittsburgh) to characterize osmolarity sensitive promoters. Sulfate Reducing Bacteria (SRB) require high salt and low oxygen conditions to grow. Therefore genetically engineering a strain of bacteria that can report osmolarity in environments such as oil pipelines and then activate a remediation circuit would be beneficial.

Firstly the promoter was tested by making a sensor with a fluorescent protein reporter. The sensor will induce expression of RFP when osmolarity is high. The sensor can be utilized to measure salt concentrations to determine if SRB can survive. The promoter can then be used to produce Dispersin B enzyme to degrade the biofilm where SRB can accumulate and ultimately, corrode the pipes. With the deadlines approaching, we shared the sequences for the osmolarity sensors with Pitt iGEM.

The two versions of the sensor, which were designed, include the native wild-type promoter sequence of Escherichia coli proU operon (REF) and the optimized Consensus promoter sequence (Lucht and Bremer, 1990), which is a synthetic sequence with mutations previously discovered to increase expression of the operon that codes for a binding protein-dependent transport system.

In this collaboration, Pitt iGEM team cloned the constructs and transformed them into MACH1-T1 cells with the help from Dr. Telmer , who is our PI. Then they proceeded to test growth of the cells in LB media with varying concentrations of sodium chloride. Finally, RFP fluorescence levels of these different cultures were measured to determine osmolarity regulated RFP expression in cells with WT and the Consensus promoters compared to the negative control.

CMUQ will transform these constructs into DH5a to account for differential expression over different bacterial strains. These studies show that promoter activity increased in conditions of high osmolarity and salt. The next step is to have the proU promoter drive Dispersin B expression to degrade the biofilms. If the cells can be concentrated at areas of high SRB growth this will be more efficient.

Since the Pitt iGEM’s project works with optical control of bacteria movement, while iGEM CMUQ’s project works with biosensors and biofilm degradation, combining these projects to optically direct the degradation of bacterial biofilms to appropriate sites will be beneficial for regulating the localization to a greater degree.

REFERENCES

Lucht JM, Bremer E. 1990. Characterization of Mutations Affecting the osmo-regulated proU Promoter of Escherichia coli and Identification of 5' Sequences Required for High-Level Expression. J Bactereol. 173:801-809.

May G, Faatz E, Lucht JM, Haardt M, Bolliger M, Bremer E. 1989. Characterization of the osmoregulated Escherichia coli proU promoter and identification of ProV as a membrane-associated protein G. Mol Microbiol 3:1521-1531.