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.

Based on above principle, one published paper has already realized synchronized oscillations under microfluidic device.[1] However, iGEM13_XMU-China can’t get synchronized oscillation on microfluidics, and that will be discussed later. Through calculating fluorescence on 96-microwell plate every 15 minutes, they got two oscillation cycles within 500 minutes (Figure 1B).

Circuit design

Based on that, we construct 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.

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 could also be formed by gene oscillator. Multiply the period by the quantity of bacteria rings, we can tell how much time has passed.

Characterization of circuit

Experiments show that bacteria could just form several rings in 48 hours. Then, no bacteria ring formed while bacteria kept spreading evenly from the inside out. As bacteria formed the rings (Figure 3A)which are quiet different from wild-type (Figure 3B).

We make sure that chemotaxis is reprogrammed successfully but not as expected. We try 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 notice that the rate of two curves kept stable expect the beginning 40 hours. So why the bacteria cannot form rings afterwards and 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.

References

1. Danino T, Mondragón-Palomino O, Tsimring L, et al. A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.

http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html