Team:FJNU-China/Design
Design
- Overview
- Garbage lid
- Bromidrosis
- Degreding enzyme
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. In this project our team aims to popularize our PLA and 2-PE to inhibit the harmful bacteria and make the environment more pleasant. After visiting the Environmental Protection Bureau for our human practice (Details can be found on our Human Practices page.), 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 be polluted by abandoned pipettetips, waste of medium, diverse biological reagent and so on. And the stink released by the garbage gives us intuitive feelings and bothered us. Therefore we try to relieve this problem using PLA and 2-PE produced by the engineered E.coli. TnaA-/-deletion strain is chosen as our engineered bacteria, which is odorless. By using this strain, we can avoid the smell of E.coli itself and keep the smell pleasant.
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 where we spent most time a day, the improperly processed garbage should be taken into consideration.
We designed our engineered bacteria in a special container: a garbage lid, PLA and 2-PE will be released to inhibit the growth in the experimental garbage and release roselike aroma at the same time.
Taking biosafety into account, we added mazEF to our circuit, which is a toxin-antitoxin module located on the Escherichia coli chromosome and some of other bacteria, including pathogens. mazF specifies for a stable toxin protein, and mazE specifies for a labile antitoxin, which antagonizes toxin protein[1]. mazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and mazE inhibits the RNase activity of mazF by forming a complex.[2]. In our project We used mazF toxin protein as our killer to ensure biosafety.
mazF, as a suicide gene, is linked 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. Upon light exposure, the light-switchable repressor LEVI is induced to form homodimers, which then bind to the operator sequence and repress the activity of promoter by blocking the attachment of RNA polymerase to the promote, represses the expression of CI repressor and initiates target gene. Upon light illumination, LEVI homodimerizes data were normalized to the mCherry expression level in dark conditions. Wavelength of activating light is 587nm, and the wavelength of emitted light is 610nm.(Details can be found on our Result page.)
We linked mazF and LightOn Switch together. Once the bacteria is revealed from the container, in other words, LightOn Switch is open, mazF will be expressed to cleavage the mRNA. And this is the reason why we use light-proof ingredient to the 3D-printed garbage lid. (Details can be found on our Application page.)
Within the laboratory, we demonstrated the efficiency of PLA sterilization. And we also obtain the most suitable concentration of 2-PE according to the data of our human practice.(Details can be found on our Human Practices page.)
Bromidrosis
Aiming at another stink caused by bacteria, the bromidrosis is also an unpleasant smell which bothers people around. Two new circuits are applyied 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, designed and construced an engineered E.coli strain with a logical gate to produce bacteriostatic PLA. Every basic component serves respective functions. dsrA, a temperature-controlled promoter from E.coli, is more active at 37 degree than 25 degree.[3] The osmotically control promoter proU shows sensitivity to the salt concentration from 0M to 0.3M.[4] We used the global-regulate protein H-NS that could specifically bind to osmotically control promoter proU as a specific repressor to inhibit the promoter downstream.[5]
When the circuits are spray on the skin, temperature reaches 37 ℃, the repressor protein LacI will be expressed to inhibit PlacI and the H-NS won’t be revealed. Thus, when people sweat, the Na+ and Cl- will induce proU resulting in the logical gate switching to “on” state.
The circuit doesn’t need to be “on” state when it is at room temperature. When temperature stays below 37 ℃, dsrA 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 on “off” state. We developed the 2-PE part as a switch that realizes the transformation from producing mode to suicide mode. TetO and TetR are common logic elements used in synthetic biology, linking with 2-PE and mazf.[6][7]
Without the induction of arabinose, TetR and mazf won’t be expressed, and production of 2-PE maintained at a normal level.
If users would like to stop 2-PE production, they only need to add a little arabinose. TetR will be expressed to inhibit TetO and mazf, causing the cell death. We currently use GFP and RFP to characterize the expression of our circuits. And we’re also trying to obtain more results. (Details can be found on our Results page.)
Degradation
We have known that the odorant acid E-3-methyl-2-hexenoicacid (E-3M2H) is an abundant and dominant human odorant. [8] 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[9] , 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.[9] AdhE2 catalyzes reduction of 3-methyl-2-acetyl-CoA to 3-methyl 2-hexenol at the expense of NADH.[10]
