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Team:Nanjing-China/qs
From 2013.igem.org
Revision as of 07:55, 24 September 2013 by GaoJian NJU (Talk | contribs)
- QS System
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First of all, we should have an understanding of how bacteria move. For E.coli, they have flagellar motor to control their movement. The direction of rotation of it is controlled by the protein CheY. WhenCheY is not phosphorylated, the flagellar motor rotates counterclockwise (CCW) and bacteria can migrate. When CheY is phosphorylated (CheY-P), it can bind to the flagellar motorprotein FliM, causing the cell to tumble and thus nonmotile. PhosphataseCheZ, dephosphorylates CheY-P and causesthe flagellum to rotate CCW. E. coli lacking thecheZ gene (ΔcheZ) cannot dephosphorylateCheY-P, and thus tumble incessantly, being nonmotile. So we only need to take control over gene cheZ to control the motility of our bacteria.
Fig.0
We would like our bacteria able to seek and destroy atrazine in the soil. To achieve this goal, a classic systemcan do the job. In this system, bacteria could move around randomly but fixed when sensing atrazine as the ribosome-switch turns on and protein CI is expressed to repress the gene cheZ. As a result, our bacteria will just assembly around the target molecules after a period of time.
Fig.1 (gene circuit of Pattern 1)
However, we can only wait for the bacteria to hang about until they find their targets. Thus it could take a long time before the bacteria assembly and the system may not be a robust design. So we shall spare no efforts to improve it.For better control over the function of our bacteria, we take the quorum sensing system into consideration. We expect some bacteria which first find the atrazine would recruit other bacteria at a distance through some signal so they move towards the target and then fixed at the source of pollution. Therefore we constructed the following gene circuit to realize it.
Fig.2 (gene circuit of Pattern 2)
This circuit is composed of three modules: an atrazine-sensing module, a recruiting module, and a brake module. We adopt the quorum-sensing system in Vibrio fischerias the basis for our design. In this system, a small-molecule acyl-homoserine lactone(AHL) synthesized by LuxI excretes as a signal and accumulates intracellularly to activate a constitutively expressed regulator, LuxR.Our atrazine-sensing module control the translation of LuxI through riboswitch which turns on when combined to atrazine. As for the recruiting module, the LuxR-AHL complex promotes cheZ transcription via promoter Plux/CI. The higher cell density is, the higher AHL concentration is, and the stronger transcription of cheZ which means greater motilityis, leading to directional movement towards bacteria which send out the signal.Finally, a repressor CI, controlled by riboswitch which turns on when combined to atrazine,represses the promoter Plux/CI and serves as a brake of the movement, resulting in bacteria fixation at source of pollution.
We establish mathematical models and use computer simulation to compare these two systemsand evaluate the improvement. In this case, we design the third system, which has the clearest logical relations, to help us better understand how the gene circuits work.
Fig.3 (gene circuit of Pattern 3)
The atrazine-sensing module is the same as the one in Pattern 2. However, the double-functional promoter Plac/CI is replaced by a series of elements. In this system, the LuxR-AHL complex promotes the expression of a repressor TetR, which represses the expression of another repressor CI. The gene cheZ is controlled by promoter Plambda, which is repressed by CI. So for those bacteria which first sense atrazine, they send out AHL signal and let the other bacteria receive it. After that, TetR is expressed to repress CI, leading the transcription of cheZ. These bacteria move towards the sender until they sense atrazine and another repressor CI, which is controlled by riboswitch, expresses to brake.
Method
1.Verification of gene cheZ in genetic level
2.Verification of gene cheZ from phenotype
3.Verification of lux system
