Team:Tokyo Tech/Genetic toggle switch to implement rewritable function

= Genetic toggle switch to implement rewritable function= In order to implement rewritable function, we tried to construct Pressure Inducible Genetic Toggle Switch (in short PIGTS). We thought two types of model - genetic circuit model and mathematical model - so as to construct PIGTS.

Genetic circuit model


1. Writable function


 * Plac is induced under 30MPa pressure


 * Express CI and GFP


 * CI represses PL and causes low LacI concentration


 * Express increasingly CI and GFP(inserted destruction tags) ⇒ Bright!!

2. Erasable function


 * PL is induced by heat


 * Express LacI and LacI represses Plac


 * Therefore, GFP expression decreases

Mathematical model
We used two differential equation models and we call them 'atmospheric pressure model' and '30MPa pressure model'. In order to implement rewritable function, we decided to use the difference of nullclines of each system so as to satisfy the following condition


 * Converge to high CI concentration state under 30MPa pressure (writable)
 * Bistable under atmospheric pressure (erasable)



IPTG assay


Objective

By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.

Result & Conclusion

GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in figure 1 and the formulation of Hill function fitting described above, the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained nP lac = 2.15.

Calculate the domain of appropriate parameters


It is known that Plac activity under 30MPa pressure is 94.0 times stronger than Plac activity under atmospheric pressure, so, &beta;30P lac  = 94.0. If 30MPa additional activity rate of PL (&beta;30P L ) or the ratio of Plac - RBS strength (&alpha;P lac ) and PL-RBS strength(&alpha;P L ) are not appropriate value, we can't construct PIGTS. So we calculated the domain of appropriate &beta;30P L and the ratio of &alpha;P lac and &alpha;P L.

We identified &beta;30P L = 1.42 by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose PL - RBS strength range from 2.4 to 7.4.