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JUDGING FORM

#Minnesota 2018

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Safety Medal Requirement JUDGING FORM

#MINNESOTA 2018

DESCRIPTION

_____Most of the mercury in the atmosphere is caused by human activities such as burning coal to produce electricity, processing taconite, and using mercury in products. Approximately 70% of current mercury comes from anthropogenic sources and 30% from natural sources. Elemental mercury vapor that occurs naturally in the environment and is released by industrial plants is oxidized to mercury(II) in the atmosphere and returned to Earth's surface in rainwater. Inorganic mercury if converted into organic forms by aquatic bacteria, and the primary methylation agents in most of these aquatic ecosystems are sulfate-reducing bacteria, which are ubiquitous in the top centimeters where most sediments transition from aerobic to anaerobic.

_____Methylmercury is one of the most toxic forms of mercury, and it will biomagnify in aquatic food chain, and it will gradually accumulate from bacteria, planktons to vertebrate organisms. Common seen species of fishes in lakes located in Minnesota, such as sunfish and bass, contain approximately 500-5000 ppm of mercury. All mercury in fish tissues/vertebrates is methylmercury and is the only form that can accumulate in human neurological tissues. Approximately 95% of fish-derived methylmercury is absorbed from the gastrointestinal tract. About 1 to 10% can be found in the blood primarily bound to hemoglobin. Methylmercury also enters the kidney and the liver and accumulates in growing hair. Methylmercury also crosses the blood-brain barrier. Mercury side effects numbness; difficulty in speaking; and loss of coordination, sight, or hearing.

_____Our team tries to provide an alternative solution to solve the methylmercury contamination problem in the lakes and streams, since the current solutions are mostly prevention and law enforcement. Previous iGEM Team Minnesota engineered mercury resistance protein into E. coli bacteria and then encapsulate these bacteria in silicon beads for water bioremediation. This award-winning idea has been experimentally proven to be effective in converting organic mercury into less toxic elemental form. However, since the engineered E. coli is encapsulated inside the silica beads to prevent unwanted proliferation in the environment, it only has a viability of up to 72 hours. The high cost, high maintenance of this system incites the question, “how can we engineer bacteria that are effective in converting organic mercury and maintain its viability for the length of the remediation process while not giving rise to any environmental risk.” Therefore, for this year’s iGEM, the goal is to expand on the previous project and develop a more enduring mercury detoxification method using engineered auxotrophic bacteria to increase the practical usability of the previously proposed system.

_____Our goal is to genetically engineer E. coli to make them capable of reducing ionic mercury to mercury substance. When the concentration of mercury ion is high the E. coli will proliferate and normally. When the concentration of mercury ion is below a certain, E. coli will not keep proliferating. Therefore, the engineered E. coli is a self-sustaining and self-maintaining solution to mercury pollution. The first step of our project was to construct a plasmid with chloramphenicol resistance as our selectable marker, this plasmid was transcribable and controlled by the LacI repressor with glutamine synthetase as the desired protein product. When IPTG, a lactose analogue, was added, GlnA was transcribed, causing glutamine synthetase to be translated. Once completed, this plasmid was transformed into a nonpathogenic E. coli strain with glnA knock out within its genome so it could not naturally produce glutamine synthase, making the E. coli dependent on IPTG for survival. Then a growth assay using glutamine-absent growth media was performed with varying concentrations of IPTG to characterize the system and colony transformation success.

_____With the previously optimized IPTG level, the degree of MerA expression was measured using a western blot. This allowed us to obtain data on the expression of Mercury Reductase and simulate the bioremediation process without using ionic mercury. The final step will be to replace the LacI repressor with a MerR regulator, which is a mercury(II)-dependent transcriptional repressor/activator that acts on gram-negative Mer operons and functions as both the positive and negative regulator. And once this final plasmid is transformed into our strain, mercury(II) dose-response growth assays and mercury concentration kinetics will be performed.

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