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Team:NCTU Formosa/Population Control
From 2010.igem.org
Wet Lab>Population Contral
Outline
In this portion of the design, we aim to achieve population control by combining a cell-signaling system (luxR/luxI) and suicidal gene for controlling the number of E.coli. The population controlling function in the engineered E.coli can warn us of high-density E. coli congregations by emitting fluorescent proteins. Green fluorescent light means the Cry circuit is functional and the population density is under control, while red fluorescent gene signals E.coli overpopulation.
1. LuxI protein (BBa_C0061) is the synthase that converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When the concentration of AHL reaches a threshold level, it binds to the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the Lux box within PtetR promoter (BBa_C0040), which then activates suicidal gene expression.
2. LuxR (BBa_C0062) is transcribed continuously by the constitutive promoter (BBa_J23106), and later works in conjunction with AHL. This design can control the concentration of LuxR protein in a steady phase, and the concentration of AHL/LuxR complex is only affected by the concentration of LuxI protein (BBa_C0061).
3. Since the expression level of LuxI protein is determined by the strength of the PtetR promoter (BBa_C0040), the population control system is determined by the strength of the PtetR promoter (BBa_C0040).
Strand D
We used RFP protein In place of the ccdB gene in our test circuit. Therefore, RFP will give us a visual approximation of how much ccdB is produced.
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.
