Team:FJNU-China/Design
Design
- Overview
- Garbage lid
- Bromidrosis
- “smell game”
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&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.
The promoter could be induced at visible light. Once the bacteria revealed from the container, mazf will express to cleavage the mRNA. And this is the reason that 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.
The circuit doesn’t need to be “on” state, when 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.)
Killer
Background
mazEF is a toxin-antitoxin module located on the Escherichia coli chromosome and that of some other bacteria, including pathogens. mazF specifies for a stable toxin, MazF, and mazE specifies for a labile antitoxin, MazE, that antagonizes MazF. (Engelberg-Kulka, H., et al. 2005)MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex.(Syed, M. A., et al. 2011).We used mazF as our killer to ensure biosafty.
Design
According to different scenarios, we designed different promoters to combine with mazF. With the humanization design, the biosafty of our projects is guaranteed.
Bromidrosis
In our scent spray, we designed a suicide switch induced by arabinose. When we don't want the engineered bacteria to work again, we can kill the engineered bacteria by spraying a spray containing arabinose. In order to ensure that our engineering bacteria do not work in other environments, we set the switch of PLA and 2-PE to salt control and temperature control. Only when the temperature reaches the body temperature condition and the salinity reaches the human sweating condition, our engineering bacteria will play a role.
Garbage can
In the trash can lid, we chose a light-controlled promoter to combine with mazF. Once the engineered bacteria leaves the dark environment of the trash can, it will be killed.
Reference
1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182. 2.https://2018.igem.org/Measurement/InterLab 3.http://parts.igem.org/Part:BBa_J61002