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Team:WLC-Milwaukee/Safety
From 2013.igem.org
Safety
Safety Mechanism Analysis in the Event of Non-lethal Horizontal Gene Transfer
The “One Ring to Secrete Them All” is designed to be a protein delivery system in the gut for both humans and livestock. We wanted to ensure that the plasmid could not be transferred to other organism in the gut or in the environment. If non-lethal horizontal gene transfer occurred we needed a mechanism for induced cellular death. In our plasmid this is achieved by genome integrated CymR repression of the T5cumate promoter in E.coli Nissle 1917, the promoter when not repressed drives the expression of Tse2 toxin. This was demonstrated by 2012 Trieste iGEM team as a safe mechanism for probiotics. If non-lethal horizontal gen transfer occurs it is likely due to native expression of CymR. This is counteracted by introducing Cumic acid, also known as 4-isopropylbenzoic acid, to the gut or area of contamination. The Cumic acid which will inhibit the CymR protein preventing T5cumate repression, stimulating Tse2 toxin expression in the organism that should not have our plasmid1. In order for this to be an effective containment technique, it had to be cheap, and nontoxic to the environment and host organisms.
We addressed the cost to ensure that remediation and containment of our plasmid was not cost prohibitive. We preformed a cost analysis based on a Sigma Aldrich quote for bulk orders on rare molecules. Using that information and the proper concentration of Cumic acid necessary for cymR inhibition (15ug/ml)2. 15 gram of Cumic acid at the cost of $43.80 would remediate 10 square meters of land to a depth of 10 centimeters by inhibiting CymR. At that rate, an entire football field to a depth of 10 centimeters could be cleansed of soil contaminated by bacteria that have our plasmid for $438.00. 10cm was established as the primary depth of bacterial growth where most bacterial proliferation occurs. Using the same cost analysis, treatment for the average human and livestock (based on cow fluid volume) would be $1.75 or $15.50 per treatment respectively. The relative cost makes this system economically viable. This is irrelevant if Cumic acid is toxic to organism.
Cumic acid has shown no physiological decrease in cellular metabolism. This demonstrates that it is not toxic at effective concentrations to organisms that do not have our plasmid in their cell2. What makes this system incredible is that it targets only organisms that contain our plasmid and selectively induces the death of those organisms. This maintains biological diversity in the soil organisms, without introducing antibiotic resistance into the soil population. This further convinced us that this mechanism for containment and treatment was ideal for our application. Further research must be done, as there is no data in-situ.
To establish this system as an effective containment mechanism we would need to perform clinical trials in humans and livestock as well as, field studies in the environment. Further research would need to be done to more accurately assess overall toxicity to humans and cattle. We would also need to assess chemical interactions in the soil to establish an effective concentration and a standard operating procedure for bio remediation. Though more research is required, this cost effective and reportedly nontoxic molecule makes our product containable.
1. Easton RW. p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and
characterization of DNA carrying the cmt operon. Journal of Bacteriology, 178(5), 1351-1362 (1996)
2. Choi YJ, Morel L, Bourque D, Mullick A, Massie B, Míguez CB. Bestowing
inducibility on the cloned methanol dehydrogenase promoter (PmxaF) of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1. Appl Environ Microbiol. 2006;6(12):7723–7729. doi: 10.1128/AEM.02002-06.
Safety according to the experts
Dr. Jarrod Erbe
Dr. James Henkel
Safety Form Summary
The one ring to secrete them all is a customizable secretion pump which uses Escherichia coli nissle 1917, a common probiotic, as the delivery organism. The genes to be expressed and secreted originated from Bacillus subtilis subtilis 168. The organism in which the parts were grown up and assembled is Escherichia coli Dh5 alpha. All of these organisms are rated at a BioSafety Level 1. We have the facilities to handle organisms of BSL 2, but since the organisms we were using were BSL 1; we operated at a BSL 1.
Each of the parts used have their origins in organisms having a BSL 1 or have no rating according to PHE.gov. The plasmid with our system is prevented from being used if transferred to other organisms due to the anti-horizontal mechanisms we have put in place. We use a CymR repressor that binds to the T5-Cumate operator which prevents the Tse2 toxin from being expressed. The purpose of this is so that if any organism were to uptake our system the Tse2 toxin would kill the organism as long as it does not have a gene for the CymR repressor. If there was any reason we would need to remove E. coli nissle 1917, the introduction of P-cumate to the system will prevent the CymR repressor from binding to the T5-Cumate operator which will induce the expression of Tse2 toxin, leading to the death of E. coli nissle 1917. The Tse2 toxin is categorized as BSL 2, but is exempt from the restrictions since it is only an effective toxin when it is inside the cell expressing it. The anti-horizontal transfer method was from parts made by the 2012 Trieste iGEM team. The complete plasmid we are creating secretes cellulase. The anti-horizontal transfer safety mechanism we use has been tested before and found successful. The strain we are using is a well-known probiotic that can be cleared from the user of the organism using p-cumate, if the organism becomes malicious.
Full Safety Forms
Safety Form- Part OneBiosafety Form- Part Two
