Experiments | Paris_Bettencourt

Project description pNasA - dpB.006

Abstract

Here, our team’s objective was to manipulate the membrane potential of engineered bacteria via electrical induction as a means of controlling gene expression.

Our approach was significantly informed by two papers, specifically (the authors of which our team has had correspondence).

Liu et al. show that *Bacillus subtilis* biofilm growth is a function of glutamate concentration present (1). To monitor glutamate levels, the authors measured the expression of nasA, a gene activated in response to glutamate starvation.

Intrerestingly, this paper also shows that glutamate uptake is inhibited when the cellular membrane becomes depolarized.

Furthermore, another study by Stratford et al. demonstrates control over membrane potential dynamics, notably depolarisation of B. subtilis and E.coli, via external electrical induction (2).

Combining these findings, our team wished to demonstrate electronic induction of the pNasA promoter by depolarising cell membranes. We hoped to demonstrate this first in B. subtilis and then in E.coli. We hoped to discover a new electrochemically induced promoter and use it then as a positive control in our promoter screening experiments.

dpB.006

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We designed a construct with the promoter pNasA and the reporter protein c99 mscarlet (dpB.006 - Bba_K4216031). Under glutamate starvation, which can be induced electrically depolarizing membranes, pNasA becomes activated and triggers the expression of the red florescent protein reporter. This florescence signal would allow for the measurement of pNasA activation at varying concentrations of glutamate and degrees of electrical stimulation.

Due to lack of time, the cloning of B. subtilis was not achieved, and we were not able to demonstrate the membrane depolarization induction of the pNasA promoter.

Figure from Liu et al.

Materials and methods

Cloning

We first assembling the construct design using Geneious Prime for integration into the model microbe bacillus subtilis. This organism was specifically chosen because of it’s use in the studies mentioned. Our strain was gifted by the ProCeD group at the Micalis Insitute by Caroline Peron-Cane. We ordered the entire synthesised Transcriptional Unit from Intergrated Dna Technologies.

We then assembled our insert with the B. subtilis shuttle backbone vector pDG268 via Gibson Assembly. Next, we cloned our construct into E. coli. After cloning, we performed DNA extraction, purification, and sent these for Sanger sequencing. The results from sequencing confirmed that we successfully constructed our desired plasmid.

Future Work

With our plasmid successfully constructed, the next step in the processes was to integrate the construct into the genome of our bacillus substilis strain. With our strain transformed, the goal was to electrically stimulate the cells, thus inhibiting glutamate uptake and thereby inducing the expression of pnasA. However, we have not yet been able to achieve results with our transformation and have not been able to iterate and trouble shoot the experimental set-up. Nonetheless, with the bulk of the work completed, there remains little left to have this component of our project become functional. Our team hopes that the work done here will be insightful and inspiring to those who wish to take the ideas forward.

References

Liu, Jintao, et al. “Metabolic Co-Dependence Gives Rise to Collective Oscillations within Biofilms.” *Nature*, vol. 523, no. 7562, July 2015, pp. 550–554, 10.1038/nature14660. Accessed 8 Oct. 2022.
Stratford, James P., et al. “Electrically Induced Bacterial Membrane-Potential Dynamics Correspond to Cellular Proliferation Capacity.” *Proceedings of the National Academy of Sciences*, vol. 19, no. 116, 18 Apr. 2019, p. 201901788, www.pnas.org/content/116/19/9552, 10.1073/pnas.1901788116.