Team:Peking/Project/Phototaxis/Design

Design

CheZ is the dephosphorylase of CheY in chemotaxis pathway. Three main characteristics actuate our consideration of CheZ for our usage. First, several experiments indicate that CheZ can effect the movement of bacteria, and increasing levels of CheZ induced by arabinose can enlarge the diameter of swarming colonies. CheZ deletion causes cells to tumble incessantly, resulting in a nonmotile phenotype in semisolid agar, and reintroducing CheZ restores cell motility. Second, CheZ is commonly used as motility-control module in synthetic biology. A combination of CheZ and the quorum sensing LuxI/R part forms stripe pattern in colony. Bacteria with CheZ under the control of an atrazine binding riboswitch proves the capability of atrazine chemotaxis. CheZ controlled by theophylline riboswitch of upstream random sequence acts as a colony reporter for orthogenesis. Furthermore, CheZ is included in some downsteam locomotive part of bacteria phototaxis such as in Halobacterium. On this basis, we decided to use CheZ to control the mobility of the bacteria.

Through rational consideration, we embark on building the circuit. CheZ can only function at a high expression level, so we linked CheZ with the strong promoter RBS B0034 and placed it in a high copy plasmid. The circuit of our phototaxis part consists two plasmids containing the Luminesensor and CheZ, respectively. Bacteria illuminated by blue light leads to repression of CheZ expression, resulting to lack of cell motility.

Figure 1. Gene circuit for Phototaxis.

After gathering principles and parameters of chemotaxis system, we then simulated the phototaxis system in a stochastic way to reflectively describe the phototaxis mechanism of our system(See Modeling Phototaxis).

Figure 2. Modeling result of phototaxis behavior.

As the modeling indicated, we designed several experiments to realize phototaxis. Firstly, we have to verify the function of CheZ. Two experiments were designed: the measurement of diameter of mobile (MG1655) and immobile (DH5 α and ΔCheZ MG1655) bacteria and the bacteria with CheZ under different strength of promoters: BBa_J23112, BBa_J23113, and BBa_J23114, whose relative expression levels of downstream gene are expected to be 1, 21, and 256, respectively. Sceondly, plates pasted polaroid of different transmittance are used to measure CheZ expression under different light strength. Thirdly, plot the bacteria in the center of the plate whose half is opaque. These experiments together are expected to have different diameter of colony or oval shape of colony to show the phototaxis.