Team:US AFRL CarrollHS/Description
Project Description
Methylophaga flavin-containing monooxygenase (bFMO) is the gene responsible for the conversion of the Indole into Isatin, which is then catalyzed into indigoid compounds. These compounds display an indigo color which can be easily detected (KAIST). Our project will focus on using bFMO as indicator to determine when toxic hexavalent chromium is present. The bFMO will be cloned into the C4-HSL circuit created by our team last year, in order to show the indigo color can be produced and easily seen. This will be done by replacing GFPa1 (green fluorescent protein) with the bFMO gene. Next, bFMO will be cloned into a construct with a T7 promoter, which we believe will be the most efficient promoter for bFMO. This construct will then be tested to ensure that the promoter will produce bFMO. Once this is done, the chrB repressor will be introduced. In order to determine when hexavalent chromium is present, a repressor, chrB, will be cloned into a construct along with the promoter, chrP, associated with the repressor. This construct will then be cloned into a pet11a backbone, which already has both the T7 promoter and rbs needed. The plasmid should then produce chrB, which will disrupt the chrP causing bFMO to not be produced. A commercial cell-free extract kit will then be used on this construct, and the T7-chrB plasmid will be added in order to make ChrB. When chromium is detected the ChrB repressor will be ‘knocked off,’ allowing the promoter to function and bFMO to catalyze into indigoid compounds, emitting a detectable indigo color.
Credit: https://2012.igem.org/Team:KAIST_Korea/Project_Background
Project Inspiration
Chromium is used often as a protective layer on most aircraft and pipelines. The chromium is added into a base layer of paint to prevent corrosion. However, consumption of chromium in even small amounts can lead to medical issues. Chromium can do a lot of damage in the environment and many of the people who use chromium still get sick. Chromium has been brought to the surface before in the case of Erin Brockovich. Hearing about these cases inspired us to create a paper-based detection system that can detect the presence of chromium. Having a product that is fast, effective, and cheap that could warn users if they should seek further action by cleaning up the chromium.
Hexavalent chromium [Cr(VI)] plays a large role in the industrial world. It is utilized as an additive in paints and coatings. Primarily, Cr(VI) is used as an anticorrosive, including use for the production of aircraft and pipelines. However, hexavalent chromium has many medical and environmental problems attributed to it. Cr(VI) is a known carcinogen, and exposure leads to multiple problems such as cancer, and affects nearly every part of the human body[3]. Even in protected industrial settings, leaks of hexavalent chromium have been reported, especially leaks into the water system. The goal of this project is to design a paper sensor part that can detect chromium in certain environments, in order to assist with the regulation of chromium exposure. This would help ensure the safety of the workers and the environment that are exposed to chromium.
Credit:
[1]Chromium III and VI. (n.d.). Retrieved from https://dhss.delaware.gov/dph/files/chromiumfaq.pdf
[2]Erin Brockovich. (n.d.). Retrieved from https://www.biography.com/activist/erin-brockovich
[3] Chromium (Cr) Toxicity: What Are the Physiologic Effects of Chromium Exposure? | ATSDR
Environmental Medicine & Environmental Health Education - CSEM. (n.d.). Retrieved from https://www.atsdr.cdc.gov/csem/csem.asp?csem=10&po=10
ASTM D1687 - 17 Standard Test Methods for Chromium in Water. (n.d.). Retrieved from https://www.astm.org/Standards/D1687.htm
ASTM D5257 - 17 Standard Test Method for Dissolved Hexavalent Chromium in Water by Ion Chromatography. (n.d.). Retrieved from https://www.astm.org/Standards/D5257.htm
HEXAVALENT CHROMIUM TESTING. (n.d.). Retrieved from http://www.caslab.com/Hexavalent-Chromium-Testing/
https://dhss.delaware.gov/dph/files/chromiumfaq.pdf