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Team:XMU-China/Project Application BlackHole
From 2014.igem.org
BLACK HOLE
—Suicide by chemotaxis
Overview
Thousands of years ago in China, people began to preserve food by curing them which was recorded in Qimin Yaoshu around 540 AD (Figure 1). Curing is any of various food preservation and flavoring processes of foods such as meat, fish and vegetables, by the addition of a combination of salt, nitrates, nitrite or sugar and it is one of the oldest methods of preserving food.[1] Table salt is the primary ingredient used in food curing. Removal of water and addition of salt to meat creates a solute-rich environment where osmotic pressure draws water out of microorganisms, slowing down their growth. Doing this requires a concentration of salt of nearly 20%. It has already been proved that 5% concentration of NaCl could inhibit the growth of E. coli. [2] However, utilizing hyperosmotic pressure to kill E. coli haven’t been fully explored in synthetic biology. This year, our team have put efforts on this topic and developed a system that will contribute to biosafety.
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Figure 1. The production of curing food. |
Circuit design
E. coli makes use of the EnvZ/OmpR system to mediate signal transduction in response to environmental osmolarity changes. EnvZ, a histidine kinase, undergoes trans-autophosphorylation, then the high-energy phosphoryl group is subsequently transferred to OmpR, a response regulator. In our system, we involved OmpR-controlled promoter (pOmpR) in (Figure 2). The expression strength of pOmpR depends upon the medium osmolarity. When medium osmolarity is increasing, the EnvZ will phosphorylate more OmpR into phosphorylated OmpR (OmpR-P), resulting in stronger expression strength of pOmpR. In our circuitry design, CheZ is upstream regulated by pOmpR (Figure 2).
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Figure 2. The schematic of osmotic-taxis design. |
Characterization of circuit
We use semi-solid medium culture with gradient concentration of sucrose to characterize the device (BBa_K1412010). And we assume that the motile ability is proportional to the moving radius. In the plot (Figure 3), when no sucrose added in, the motile ability is the weakest. The motile ability keeps growing as the concentration of sucrose increases from 0 to 4%. Then the motile ability goes down slightly as the sucrose concentration increased from 4% to 10%, but the ability is still stronger than that at concentration 0. We can draw a conclusion that our device is working as expectation, the motile ability goes down (4%~10%) because of the inhibition from hyperosmotic pressure.
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Figure 3. The plot of moving radius versus sucrose concentration. The four curves were measured after 10 h, 11 h, 12 h and 16.5 h respectively. |
Based on the characterization, we spotted hyperosmotic pressure spot and reprogrammed CL-1 spot on semi-solid medium culture as Figure 4 shows. The concentration will decrease with the increase of the distance away from hyperosmotic pressure spot. As osmotic pressure is proportional to the medium concentration. The moving tendency of reprogrammed CL-1 will orient to the hyperosmotic pressure spot. Even at the inhibiting osmotic pressure, the motile ability is still stronger than that without any inducer. So that reprogrammed CL-1 may even swim towards the high-osmotic site and die. The killing mechanism is just like the black hole. When the bacteria move into the “event horizon” where the osmotic pressure reaches to the critical value named the killing osmotic pressure, the bacteria can’t go out of the border and be killed finally.
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Figure 4. Schematic of killing bacteria by black hole. |
We draw a horizontal line with 10% sucrose and erect line with water, and spotted cells on the cross (Figure 5). Culturing for 48 hours, we observed that reprogrammed E. coli has significant orientation to high concentration line. As high concentration sucrose generates high hyperosmosis, it has proved that CL-1 has the tendency swimming to high osmotic pressure.
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Figure 5. Drawing horizontal line with 10% sucrose and erect line with water. Spotting cells on the cross. Two plates are parallel experiment. |
Meaning
The sources (such as NaCl and sucrose) to create hyperosmotic pressure are cheap, accessible and environmentally friendly, while antibiotics is expensive and have a bad effect on environmental microbiology because of drug resistance. If our black hole system could be fully developed, it will reduce the barriers to microbiology research especially for the scientists in poor countries.
References
1. http://en.wikipedia.org/wiki/Curing_(food_preservation)
2. SUN Z, WANG J, LU M, et al. The Inhibitory Function of NaCl to a Few Common Bacteria [J][J]. Sea-Lake Salt and Chemical Industry, 2007, 1: 004.
