Difference between revisions of "Team:FJNU-China/Design"
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[7] Saini M, Chen M H, Chiang C J, et al. Potential production platform of n-butanol in Escherichia coli[J]. Metabolic Engineering, 2015, 27:76.</br> | [7] Saini M, Chen M H, Chiang C J, et al. Potential production platform of n-butanol in Escherichia coli[J]. Metabolic Engineering, 2015, 27:76.</br> | ||
[8] Anu Jose Mattam and Syed Shams Yazdan. Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols. Biotechnol Biofuels. 2013; 6: 128.</br> | [8] Anu Jose Mattam and Syed Shams Yazdan. Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols. Biotechnol Biofuels. 2013; 6: 128.</br> | ||
| − | + | [9]Engelberg-Kulka, H., et al. (2005). "mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria." J Cell Sci 118(Pt 19): 4327-4332.</br> | |
| + | [10]Syed, M. A., et al. (2011). "The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system." J Bacteriol 193(5): 1122-1130. | ||
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Revision as of 09:10, 16 October 2018
Design
- Overview
- Garbage lid
- Bromidrosis
- Killer
Overview
As we mentioned in our background, there is a number of microorganism in our daily life which could cause disease infection and give off stink. This year our team aims to popularize our PLA and 2-PE to change the situation caused by harmful bacteria. After we went to Environmental Protection Bureau for our human practice, we thought the condition in garbage can is much more complicated than we imagine. So we temporarily focus on the microbe pollution in our laboratory, which may cause by abandoned pipettetips, waste of medium, diverse biological reagent and so on. And the stink releases by the garbage gives us intuitive feelings and bothered us. After we learnt about the characterization of PLA and 2-PE, we would like to give this problem a nice try.
Garbage lid
Irregular experimental operation is common but serious somehow, which could cause infection of disease. The pointed part of used pipettetips and different biological reagent are breeding grounds for bacteria. As the laboratory is the place we went to the most frequently and the garbage usually is not properly processed, this problem needs attention.
We designed our engineered bacteria in a special container, a garbage lid, to deal with this situation. We put it in the laboratory baker, constitutive promoter will bring PLA and 2-PE into play, to inhibit the growth in the experimental garbage and release roselike aromatic at the same time. Taking biosafety into account, we added toxin protein, mazf, as a suicide gene, linking with light-control promoter to prevent our engineered bacteria revealing to the outer environment than the garbage lid.
We completed the experimental collaboration with ECUST on the LightOn Switch. The promoter could be induced at visible light range. Wavelength of exciting light is 587nm, and wavelength of emitted light is 610nm.(Details can be found on our Collaborations page.)
Once the bacteria revealed from the container, in other words, LightOn Switch is open, mazf will express to cleavage the mRNA. And this is the reason why we use light-proof ingredient to 3D-printed our garbage lid. . (Details can be found on our Application page.) Within the laboratory can be controlled, we demonstrated the efficiency of PLA sterilization. And we also got the most comfortable concentration of 2-PE according to the data of our human practice.(Details can be found on our Human Practices page.)
Bromidrosis
After came up with the idea to solve the outer environment, we found the human environment is also interesting. Aiming at another stink caused by bacteria, the bromidrosis is also an unpleasant smell which bothers people around. Two new circuits are applying to the goal of controlling bromidrosis. When people sweat, there is specific salt concentration and temperature on the skin. We choose this feature as a starting point, designing a logical gate to produce PLA. Every basic component serves respective functions. dsrA is a temperature promoter from E.coli, more active at 37 degree than 25 degree.[1] The osmotically control promoter proU provides sensitivity to the salt concentration from 0M to 0.3M.[2] And global-regulate protein H-NS which could specifically bind to osmotically control promoter proU, we use it as a specific repressor to inhibit the promoter downstream.[3]
When the circuits on the skin, temperature reaches 37 degree, the repressor protein LacI will be expressed to inhibit PlacI, the H-NS won’t be revealed. Then, once people sweat, the Na+ and Cl- will induce proU, the logical gate became “on” state.
Another situation is that the circuit doesn’t need to be “on” state, if it store at room temperature. Temperature stays below 37 degree, the temperature promoter will not work. The function of PlacI BBa_R0011 has become a constitutive promoter. The global regulate protein H-NS will be expressed to inhibit proU. Then the logical gate will be “off” state. Then, the 2-PE part, we developed a switch which realize the transform from producing mode to suicide mode. TetO & TetR are common logic elements used in synthetic biology, linking with 2-PE and mazf.[4][5]
Without the induction of arabinose, TetR and mazf won’t be expressed, and production of 2-PE maintained at a normal level.
If the user would like to stop the 2-PE production, adding tiny concentration of arabinose will be able to achieve. TetR will be expressed to inhibit TetO and mazf caused cell death. We currently use GFP and RFP to characterize the expression of our circuits. And we’re also trying to get further data results. (Details can be found on our Results page.)
Degreding enzyme
In another way of thinking, we have known that the odorant acid E-3-methyl-2-hexenoicacid (E-3M2H) is abundant and quantitatively dominant human odorant. [6] Therefore, we tried to eliminate such unpleasant axilla odors in an innovative method by degrading the odorant acid. Referring to potential production platform of n-butanol in E. coli [7] , we designed a combined part to degrade E-3-methyl-2-hexenoicacid (E-3M2H).
This gene cluster expresses three enzymes, of which atoD and atoA from E.coli and adhE2 from Clostridium acetobutylicum ATCC 824. The function of atoDA mediates reductive conversion of 3-methyl 2-hexenoic acid to 3-methyl-2-acetyl-CoA with acetyl-CoA as the CoA donor, for fatty acid activation and its subsequent degradation.[7] AdhE2 catalyzes reduction of 3-methyl-2-acetyl-CoA to 3-methyl 2-hexenol at the expense of NADH.[8]
