Team:MichiganState/Design/Biosensor

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Project Design

General Anoxic HGT Biosensor Bioreactor Design

A potential issue posed by this design is the buildup of sedentary metabolic waste products in the microbial environment. Increasing concentration of methane metabolism byproducts (i.e., formate) causes the acidity of the environment to rise. This pH drop may slow the methanotrophic biofilm’s functioning, or even stop cell growth and division. To monitor formate levels, a fluorescent biosensor was engineered.

A common bacterial receptor molecule for formate is the formate hydrogenlyase (fhlA), a transcriptional activator of the hyc and hyp operons. ¹ Generally, when formate binds to fhlA there is a conformational shift that allows the activator to complex with RNA polymerase. The activator guides the polymerase to bind the formate dehydrogenase (fdhF) promoter sequence. This polymerase binding to the promoter, in turn, initiates transcription of downstream genes. A generalized cartoon illustration of this process can be seen in Figure 1.

Figure 1. Simplified depiction of positive transcriptional activation by fhlA

This fdhF promoter DNA will be synthesized and cloned into plasmid pRL814-GFP (obtained from Escherichia coli strain WM3064) using Gibson assembly to create the first biosensor construct; see Figure 2. This plasmid was previously engineered to contain the coding sequence for a common signaling molecule implemented in biosensors: green fluorescent protein (GFP). Transcription and subsequent translation of GFP will cause cells to fluoresce, emitting the frequency of green visible light. By fusing the coding sequence for GFP to the fdhF promoter, transcription of GFP may be controlled by formate binding to the fhlA activator. Initial assays to determine the biosensor strength will be performed in a continuous plate reader which measures fluorescence levels of GFP (excitation/emission 475/509 nm) in 96-well plates containing cells inoculated in media of varying formate concentration. The first construct to be assembled, shown below, was designed using Benchling. ²

Figure 2. Initial Biosensor Plasmid Construct containing the Formate Dehydrogenase Promoter Sequence

The construct should function properly in E. coli, as the E. coli genome naturally codes for fhlA. Methylotrophs, however, lack the proper genomic DNA to produce this activator. For the biosensor to function in the methylobacterium, the fhlA genes must also be engineered into the plasmid. From the isolated genomic DNA of E. coli WM3064, the fhlA sequence will be extracted. Gibson assembly will then be performed to remove lacI from the previous construct and insert the fhlA sequence in its location. This final construct, which should be functional in methanotrophs, is pictured below in Figure 3.

Figure 3. Final Biosensor Plasmid Construct containing the Formate Dehydrogenase Promoter Sequence and the Formate Hydrogenlyase Coding Sequence

References

¹Rossmann, R., Sawers, G., & Böck, A. (1991). Mechanism of regulation of the formate‐hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon. Molecular microbiology, 5(11), 2807-2814.

² The Life Sciences R&D Cloud. https://www.benchling.com/ (accessed May 2019).