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The "Elf": Background

Note: The "Elf" was a part of our project "A Tale of Aptamers: Ghost and Elf" until regional jamboree in early October. As we think the Bacterial Ghost should be more valuable and applicable, we decided to focus on the Bacterial Ghost and lay aside the Atrazine "Elf". Therefore we changed the title and contents of our project. The previous work (section "Elves' Cooperation") is moved here.

Declare: The "Elf", namely the atrazine seek and destroy system, is an original work of ZJU-China iGEM Team.

Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) has been long used as a sort of herbicide since synthesized decades ago. For its feasibility and low price, many countries including U.S. are employing atrazine at ease in weed clearance. By blocking the plastoquinone-binding protein in photosystem II, atrazine selectively suppresses the growth of targeted weeds in the field and concurrently, minimizes the negative effect on major crops. The death of unwanted plant is a result of starvation and oxidative damage due to the breakdown in the electron transport process. Thus, the biomass accumulation of economic crops increases and that brings us a higher yield.

However, atrazine functions as a double-edged sword, for recent reports on it are questioning its safety by presenting biological evidence from the following perspectives. Ecologically, atrazine reserves itself steadily in the soil. Because of the hydrologic cycle, it contaminates water as well. The pollution is pretty hard to be physically or chemically disposed of, as atrazine owns a good solubility and stability in water. In the research of amphibians, it is reported that a potential cause of birth defects, low birth weights and menstrual problems in frogs. For human, a compromise on endocrine system, possible carcinogenic effect and uncertain connection to low sperm level are claimed as a result of incorporating atrazine. As a result, atrazine is banned by Europe Union since 2004. The blocked use of it prevents the contamination in the future. Nonetheless, previous damage is not involved in the enactment, so the problem is still in a desperate need to be solved.

A recent paper published in 2010 caught our attention in which the researchers engineered E.coli by imparting motility and atrazine-degradation function to it. The bacteria are able to sense the atrazine gradient and swim against it. While approaching the center of contamination, an atrazine-degradation enzyme starts to function to eliminate atrazine.

The ‘plants verses zombies’ model is fascinating, however, several problems remain. Once a wave of bacteria crowd into the ‘battlefield’, atrazine along the way is degraded and the gradient no longer exists to renew the next round of attack. Under the microscope, the pattern goes like a superimposition of concentric rings of different diameters. In addition, the atrazine-degradation enzyme gene is environmentally detrimental, instead of plugging the gene into the genome, previous research adopt plasmids as the carrier. Horizontal gene transfer is a putative problem if the engineered cells are put into practice. Finally, the modified bacteria have unknown ecological niche. That is to say, the innocent E.coli are possibly substituted by our engineered ones, which is a nightmare for everyone – every human being on the earth and every innocent E.coli in the earth. To solve the aforementioned problems, we’ve developed a novel method in which two kinds of engineered bacteria are involved. The first group sense and move against the atrazine gradient. Let’s call them detectors. At the same time, detectors release AHL, a quorum-sensing related substance which is in charge of attracting the second group of bacteria. The second group, we call them cleaners, move toward detectors and clear atrazine in their way. In order to prevent the atrazine-degradation bacteria from invading other species, we equip cleaners with holin-antiholin circuit which blocks horizontal gene transfer. For similar ecological reasons, a ccdB circuit is added on the circuit of detectors to control the cell density within a certain range.

To sum up, our design is a ‘5E’ system as follows:

1-2. Efficient and Exhaustive clearance. We’ve proved that our design helps improve the clearance both spatially and temporally. Concentric pattern is eliminated and that the novel atrazine degradation method is proved exhaustive. We also test the dynamic parameters in the timeline. It is observed that atrazine can be removed within few hours, which supports the idea ‘efficient’.

3. Eco-friendly pattern. With the aid of ccdB circuit and holin-antiholin circuit, we secure the environmental safety with two methods – density control and prevention of plasmid leakage.

4. Elaborate design. We are among the first to raise a novel theoretical model in which quorum sensing and motility elements are involved. The collaboration between two sorts of bacteria is a promising approach to learn the behavior of bacteria, and to be practically used in agriculture.

5. Easy to store. To postpone the expiration date, we employ a chemical method to encapsulate our cells with a specific compound. The shell enables our product being stored at room temperature for months.

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