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Team:NCTU Formosa/Population Control
From 2010.igem.org
Wet Lab>Population Control
Outline
In order to make an environmentally safe insecticide, the Mosquito Intelligent Terminator (MIT or Terminator) incorporates a genetic circuit with LuxI/LuxR device for controlling the population size of the terminator. When the Terminators are released in the environment, the Terminator population can be self-maintained by our system thus a surplus will never exist using. In addition, we can manually kill all Terminator's by spraying AHL to induce self-destruction.
Component Descriptions
1. In strand C, Ptet promoter (BBa_R0040) express the downstream genes, LacI (BBa_C0061) and LuxR (BBa_C0062) constitutively at room temperate.
2. LuxI protein (BBa_C0061) is the synthase that convert S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When the concentration of AHL reaches a threshold level, it binds the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the Lux box within Plux (BBa_R0062) promoter in strand D to activate transcription of the downstream ccdB gene (BBa_ K145151) and red fluorescent protein (BBa_ E1010).
Principle and Mechanism
The population control system has three statuses:
1. Reproduction status at 37°C in laboratory
2. The self-maintain status.
3. The self-destruction status by AHL administration
1. Reproduction status at 37°C in laboratory
When bacterium grows in 37°C, the constitutive promoter transcript tetR repressor inactivates the promoter Ptet (BBa_R0040) on strand C, subsequently LuxI andLuxR protein are not transcribed. Accordingly, the population control system is not activated during bacterium growth. This design allows us to amplify the terminators without any potential limiting factors.
Fig. 2:The reproduction status when bacterium grows at higher than 37°C.
2. The self-maintain status
When the Terminators are released in the environment at room temperature, the promoter Ptet promoter transcribes the LuxI and LuxR protein. LuxI protein converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When external bacteria aggregate, the concentration of AHL will rise pass threshold level and resulting the formation of AHL-LuxR complexes. This complex then becomes a translation factor to Plux (BBa_R0062) promoter and expression of downstream ccdB gene is activated. Accumulation of ccdB protein result in ccdB production and subsequent suicide. Therefore, when the population size of E. coli rises pass the threshold level, ccdB proteins are induced by AHL to restrict the population size (Fig. 4).
Fig.3: The self-maintain status when bacterium grows at less than 37°C.
Fig.4: The simulation result of the self-maintain status. When the population size of E. coli rises to the threshold level, ccdB proteins are induced by AHL to restrict the the population size.
3. The self-destruction status by AHL administration
After Mosquito Intelligent Terminators have served its purpose, we can manually kill all Terminators by spraying AHL to induce self-destruction. LuxR is a constitutively expressed protein that can bind AHL, which together form a complex activating Plux. ccdB's are produced after Plux is activated and when in combination results in E. coli self-destruction.
Fig.5: The self-destruction status induced by AHL administration.
Procedures
(I) Strand C (BBa_ K332021)
1. Strand C is synthesized from three fragments – (1) Ptet (2) RBS+LuxR (3) RBS+LuxI+double ter sites.
2. All of them are in the form of plasmids, so we digest them by appropriate restriction enzymes.
3. We first combine the last two fragments by ligation. The procedure is as follows:
(1)Add the respective digestion products, vector plasmid, buffer, ligase to ddwater
(2)React at 16°C for 30 minutes
(3)Inactivate the ligase by incubation it at 80°C for 20 minutes
(4)Store the products at 4°C
4. The products of ligation are then transformed into competent cells. The procedure is as follows:
(1)2µl plasmids(ligation products) are added to 33µl competent cells
(2)Shake gently for one second, and cool it on ice
(3)Incubate it in 42°C water bath for 45 seconds
(4)Spread the liquid to plate which corresponding antibiotics have been added to
(5)37°C overnight
5. The next day we do a colony PCR for the transformed cells and have the PCR products electrophoresis to make sure that the two fragments have been combined correctly.
6. Now we separate the plasmids(RBS+LuxR+RBS+LuxI+double ter sites) from E. Coli, and digest it with restriction enzyme.
7. The first fragment (Ptet) is then combined with the above product by ligation, and transformed into competent cells by the same we have done early.
8. Because the length of Ptet to the remain segment is relatively too short, we cannot confirm whether they have combined correctly by just do an electrophoresis of the colony PCR products. If the electrophoresis result seems like that they have combined, we then send the product to sequencing.
(II) Strand D (BBa_ K332022)
1. Strand D is synthesized from two fragments – (1)Plux (2)RBS+mRFP+double ter sites
2. The plasmids are digested into corresponding products used for ligation.
3. Combine the two fragments by ligation, the protocol is the same as we have done for Strand C.
4. Transform the products from step 3 ligation.
5. Colony PCR for the transformed cells and have the PCR products electrophoresis to make sure that the fragments have been combined correctly.
6. If the electrophoresis result show that the length of Strand D is as expected, we then send the products for sequencing.
