Team:USTC-China/Project/results
From 2011.igem.org
![](https://static.igem.org/mediawiki/2011/3/31/%E6%A0%87%E9%A2%98%E6%A0%8F%E6%9C%80%E7%BB%88%E6%8E%A8%E8%8D%90%E6%9C%89%E5%AD%97%E7%89%88.png)
Experiment results of the Basic Design
and Discussion
and Discussion
1.Verification of ΔcheZ strain
2.Verification of the function of the constructed Aptamer-cheZ part
3.Verification of the original Toggle-switch from PKU
4.Verification of the modified Toggle-switch
5.Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch
6.Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch
Verification of ΔcheZ strain
The size of colonies of E.coli strain RP1616 was much smaller than that E.coli steain RP437 under the same circumstance and after same period of incubation time(about 10h), and the result is shown in Figure1. In colony PCR using the primers of cheZ gene following , RP437 absolutely has much more outcomes than RP1616 and we conclude that RP1616 is actually a ΔcheZ strain.
Verification of the function of the constructed Aptamer-cheZ part(protocol1)(protocol2)
From the results shown in Figure2. We can be sure that the Aptamer-cheZ part actually works effectively, especially on 0.3% Semi-solid medium.
Verification of the original Toggle-switch from PKU(protocol)
By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 8:25. It means the original Toggle Switch is not fit for our purpose, so we modify the original by luxPR-cI device.
Verification of the modified Toggle-switch (protocol)
By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 6:1. It is useful to our project design.
Test of Incorporated Aptamer-cheZ part into one side of the original Toggle-switch (protocol1) (protocol2)
According to the results above, by using this device we can not make the reprogrammed bacteria be divided into two different states effectively, and because of the bias to the green fluorescence and moving towards the high concentration of the theophylline, the bacteria trending to stay are very rare.
Test of Incorporated Aptamer-cheZ part into one side of the modified Toggle-switch (protocol)
Figure9 clearly display the dividing line between the reprogrammed bacteria at two different states, and the bacteria mainly express GFP indeed moving towards the high concentration of theophylline. This means the modified Toggle-switch-Aptamer-cheZ actually work as our design.
Figure10.The SDS-PAGE result of the cheZ protein
Figure11.
Figure10 shows that, the bacteria with RFP do not express cheZ, and the expression of cheZ of the bacteria with GFP increases along the theophylline gradient.
Discussion
The experiment result mainly depend on the the random fluctuation of expression of the modified Toggle Switch device, so this device has a certain probability to switch from one state to the other, then it may lead to stopping of the moving bacteria like colony2(Figure12.).
To weaken this randomness, we may use the quorum sensing to control the moving of the reprogrammed bacteria, and we construct a model to simulate this proposal by computer (Modeling).