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Team:XMU-China/Project Application OscillationTimer
From 2014.igem.org
OSCILLATION TIMER
--Ameliorate last project by chemotaxis
iGEM13_XMU-China has tried to construct oscillation system by standard biobricks. The synchronized oscillation system used in that study(Figure 1A) is based on the quorum sensing machineries in Vibrio fischeri and Bacillus thurigensis. Three identical luxI promoters are in charge of luxI (from V. fischeri), aiiA (from B.thurigensis) and gfp genes separately. The LuxI synthase generates an acyl-homoserine-lactone (AHL), which can spread across the cell membrane and mediate intercellular coupling. AHL then binds to LuxR produced intracellularly, and the LuxR-AHL complex would activate the luxI promoter. AiiA catalyzes the degradation of AHL as the negative feedback in the circuit. Therefore, both the activator AHL and the repressor AiiA of the network are activated by the luxI promoter simultaneously.
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Figure 1A. Schematic of the oscillation based on quorum sensing system.1B.Two oscillation cycles were observed within 500 minutes by microplate reader. |
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Based on above principles, one published paper has already realized synchronized oscillations under microfluidic device [1]. However, iGEM13_XMU-China can’t get synchronized oscillation on microfluidics, which is discussed in P SYSTEM. Through calculated fluorescence on 96-microwell plate every 15 minutes, it got two oscillation cycles within 500 minutes (Figure 1B).
Circuit design
Based on that, we constructed our circuit by replacing gfp with cheZ (Figure 2). As the expression strength of cheZ is oscillatory fluctuating, the motile ability will change periodically. Cells will have the strongest motile ability at wave crest while even be non-motile at wave trough. Thus, periodical change of motile ability leads to periodical change in swimming velocity. At non-motile period, cells will aggregate together leading to the formation of growth-ring-like patterns which could be distinguished by naked eyes.
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Figure 2.Schematic of the growth-ring formation circuit. Derived from quorum sensing oscillator by replacing GFP with CheZ. |
Many trees in temperate zones make one growth ring each year, with the newest adjacent to the bark. We can tell a tree’s age by counting the number of growth rings. Analogously, bacteria rings formed by gene oscillator can tell us how much time has passed through the quantity of bacteria rings.
Characterization of circuit
Experiments showed that bacteria could just form several rings in 48 hours. Then, no bacteria rings formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (Figure 3A) which are quite different from wild-type (Figure 3B), we can conclude that chemotaxis is reprogrammed successfully, however, not as expected.
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Figure 3A. Bacteria rings formed by CL-1 with above oscillation circuit, it has several bacteria rings in the beginning.3B. Bacteria rings formed by wild-type E. coli (CL-M). It is different from the right picture. |
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Therefore, we tried to make use of the grown time (as X axis) and radius of grown (as Y axis) to draw a curve to see whether the amplification rate of chemotaxis radius are equal over a period of time (Figure 4A, 4B). We noticed that the rate of two curves keep stable expect the beginning 40 hours. So why the bacteria cannot form rings afterwards but the grown rate becomes stable? We thought that there must be something wrong with the negative feedback in the circuit. Circuit can’t generate enough feedback to repress the chemotactic ability so that swimming speed keeps constant which is actually maximum speed. Hence, combining the experimental results of iGEM13_XMU-China with ours, we are still trying to find out why the oscillation could just keep several periods, and we got a reasonable conclusion that may help us make it clear.
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Figure 4A. The plot of chemotactic diameter versus time. Cultivated CL-1 on semisolid culture medium with chloramphenicol and tetracycline (halve concentration) and made use of the grown time (as X axis) and chemotaxis diameter (as Y axis) to draw a curve, the curve show that the diffusion rate is stable. 4B. A parallel experiment to Figure 4A |
When we were improving the project of iGEM13-XMU-China, we creatively put forward an assumption to a problem mentioned in P SYSTEM which has been ignored for a long time.
Want to see more applications: Promoter Yardstick,Black Hole, Aptamer Key-Lock.
References
