Team:TokyoTech/Experiments
Main Experiments
Turing pattern is formed by the interaction of two substances with different rate of diffusion. One of them works as an activator and the other one works as an inhibitor. The activator promotes its own expression and expression of the inhibitor, and the inhibitor is required to suppress the expression of the activator.
PAI1 produced by LasI gene acts as an activator, and PAI2 produced by RhlI gene acts as an inhibitor.
We will look at the specific genetic circuit function over time.
This genetic circuit begins to function when DsRed is expressed over time.When DsRed is expressed, the spot shows red fluorescence.
Then, regulated by the same promoter as DsRED, LasI and RhlI produced PAI1 and PAI2 respectively. As mentioned above, they have different rate of diffusion, in which PAI2 diffuses at a higher rate compared to PAI1.
When diffusion occurs, PAI1, together with LasR activate itself and RhlI expression by acting on pLas-OR. This acts as an activator in the Turing pattern formation. Expression of DsRed are also promoted in the same process.
In addition, PAI2 promotes the expression of GFP and CI, while inhibiting the expression of itself and LasI/DsRed by inhibiting the function of the promoter containing the binding site. This acts as an inhibitor in the formation of Turing pattern.
At this point, PAI2 is also present in the range where PAI1 diffused, but because PAI1 also inhibits the binding between PAI2 and RhlR, so PAI2-induced GFP expression is suppressed in the range of diffusion by PAI1.
Also, the pattern can be manipulated by introducing other control mechanism into the promoter where RhlR / PAI2 complex binds to.
We have considered a hybrid promoter (BBa_K3259000) that can be controlled by PAI2 and blue light, and also a hybrid promoter (BBa_K3259001) that can be controlled by PAI2 and temperature.
With the presence of PAI2, when BBa_K3259000 is not exposed to light, it will bind to Escherichia coli -derived BluR protein, thereby suppressing gene expression. However, when it is exposed to blue light, the BluR protein will bind to the BluF protein and can no longer inhibit gene expression.
By doing so, the expression of CI and GFP becomes more active when we exposed it to blue light compared to when there is no light, and the size of red spot becomes smaller.
In addition, even with the presence of PAI2, the structure of promoter BBa_K3259001 are not stable at about 30 degrees, and gene cannot be expressed. However, when it is placed at a lower temperature of about 18 degrees, the structure becomes stable and gene expression is activated.
By using this hybrid promoter, the size of red spot will become smaller at low temperatures.
In this project, we are working on ways of âcontrollingâ patterns, but in the future we aim to create a system that can be âcontrolledâ by environmental factors.
