In literature, the development of biosensors for detecting heavy metals is extensively discussed and used as a tool to combat environmental degradation. As the AshesiGhana team, we considered developing biosensors for gold prospecting thereby saving time, reducing expenditure, and mitigating environmental degradation. We engineered bacteria to sense the presence of several pathfinders (Au+ , As3+, Fe2+) and to give a color indicator that’s either blue, yellow, or pink respectively. This will simplify, expedite, and lower the cost of gold prospecting. We chose our chassis carefully to follow a sustainable and environmentally friendly strategy, ensuring maximum growth and functionality while upholding environmental safety.
We chose to use Escherichia coli (E. coli) because it is a cornerstone for many essential findings in molecular biology and other areas of cell physiology. The fast growth in chemically defined media, relatively affordable culture media, and extensive knowledge of its genetics and genomics, among others, were important factors in our decision-making [1]. In addition, the presence of the ars operon in E Coli makes it suitable for the detection of arsenic [2], thus, making E Coli a great chassis for our project.
It is possible to convert a transcriptional module into a quantifiable signal, achieved by a reporter gene, for instance, a chromoprotein (CP), which is a colored protein that can be seen with the naked eye. CPs have some benefits over fluorescent proteins (FPs), including dark colors in ambient light that allow for low-cost, instrument-free examination [3]. Hence, CPs were used as reporter genes for our project.
Gold Sensing ModuleThe golTSB from the Salmonella enterica serovar typhimurium is regulated by Au ions to provide gold resistance to the bacteria. In one experiment, this regulon was engineered by attaching a promoterless reporter gene lacZ “downstream of the golB open frame as a transcriptional fusion” (i.e. golTSB::lacZ) [4]. Recent research has shown that GolS promotes the transcription of the golTS operon and golB, a tiny neighbouring gene that codes for a putative metal-binding protein when there is Au ions present [5]. golB is a gold-binding protein to ensure that gold ions do not move freely in the cell.
Also, there are regulatory promoters (PgolS & PgolB) controlled by golS. In order words, golS induces transcription from these promoters once golS is activated upon binding a gold ion [6], illustrated in figure 1. PgolTS works as a constitutive promoter ensuring the production of golS protein, in the presence of Au+, the golS binds with the gold ions stopping the inhibition of PgolB, hence transcription. The reporter gene is expressed by attaching a promoterless amilCP to produce that blue colour even as the golS proteins bind with gold ions in the cytoplasm (Figure 1).
Arsenic Sensing ModuleUsing an inducible promoter comes with shortcomings such as high leakage, low induced fold change, and poor sensitivity. Hence, the team utilized a constitutive promoter regulated by the ArsR regulatory gene. Given that the constitutive promoter is always active independent of the transcription factors, in the absence of arsenite (As3+), ArsR binds to the RBS to repress Pars promoter transcription. However, in the presence of As3+, the arsR protein binds with the arsenite, triggering the reporter gene (amilGFP) expression and resulting in the yellow glow of the bacteria.
Iron Sensing ModuleThis module consists of a constitutive promoter Pcat which is by default independent of the transcription factor. The FUR protein acts as a repressor for the PAceB and is always produced. In the absence of Fe2+, the PAceB is turned on, allowing for the transcription, resulting in the TetR binding repressing the Ptet, inhibiting further transcription, therefore the reporter gene is not expressed. However, in the presence of Fe2+ , the FUR protein binds with the ions, repressing the PAceB hence the TetR protein won’t be produced and Ptet is turned on allowing for the reporter gene eforRed to be expressed, giving out a pink glow.
Kill Switch ModuleTTo ensure no escape from the predetermined surroundings, the kill switch gene is crucial for eradicating the bacteria that might have escaped from our device . A T4 endolysin and a UV promoter make up our kill switch. The UV promoter is an inducible promoter that is regulated by UV irradiation. When the peptidoglycan layer of the E. coli is degraded, the T4 endolysin can release lysozyme, which causes cell lysis and ultimately kills the bacteria, preventing it from interacting with other species in the environment.
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[2] A. Carlin, W. Shi and S. Dey, "The ars Operon of Escherichia coli Confers Arsenical and Antimonial Resistance," Journal of Bacteriology, vol. 177, no. 4, p. 981–986, 1995.
[3] L. P. Bao, N. K. Menon, J. Liljeruhm and A. C. Forster, "0003-2697/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Overcoming chromoprotein limitations by engineering a red fluorescent protein," Analytical Biochemistry , vol. 611, no. 113936, 2020.
[4] C. M. Zammit, D. Quaranta, A. J. Z. Shane gibson, J. B. Christine ta, R. y. Lai, g. Grass and F. Reith, "A whole-cell Biosensor for the Detection of Gold," Plos One , vol. 8, no. 8, 2013.
[5] S. K. Checa, M. Espariz, M. E. P. Audero, P. E. Botta, F. C. Soncini and S. V. Spinelli, "Bacterials sensing of and resistance to gold salts," Molecular Microbiology, vol. 63, no. 5, pp. 1307-1318, January 2007.
[6] T. Hsiao, "iGEM Parts," 26 October 2010. [Online]. Available: http://parts.igem.org/Part:BBa_K310009.