Team:Tokyo Tech

2. Pressure induction
Introduction

Construction



Result -activity of tet promoters- 



3. Touch display
Touch display (plan)





Prototype touch display





Result ~ E. coli in the touch display ~



                   

4. Low pressure-inducible promoter
It is known that lac promoter is induced under 30 MPa (T. Sato et al., 1995). However, 30 MPa is too high to push with the fingers. Therefore, we tried to develop low pressure-inducible promoter.

Methods

We sorted fluorescent (A) and non-fluorescent bacteria (B) with a flow cytometer. Then, we analyze these base sequences. Therefore, fluorescent bacteria have no mutation in CAP binding site, -35 or -10.
 * A have mutations in LacI binding site or non-functional DNA.
 * B have mutations in CAP binding site, -35 or non-functional DNA.

We have successfully demonstrated that it is possible to collect promoters desired functions by PCR random mutagenesis and screening with a flow cytometry. This results indicate that we can screen low pressure-inducible lac promoter mutant with this methods.

5. Write/Erase cycle


While we can implement write-function, we want to implement additionally erase-function and memory-function. Erase-function enables us to erase the painted picture, and memory-function enables us to keep the picture after we stop induction. We call these functions "Write/Erase cycle". In order to implement Write/Erase cycle, we tried to construct genetic toggle switch.

Genetic toggle switch to implement Write/Erase cycle



 * 1) Write-function
 * 2) Plac is under 30 MPa pressure.
 * 3) Plac expresses CI and GFP.
 * 4) CI represses PL and decreases LacI expression.
 * 5) Low LacI expression increases Plac activity. ⇒ Bright!!
 * 6) Erase-function
 * 7) The heat activates PL.
 * 8) PL expresses LacI.
 * 9) LacI represses Plac.
 * 10) Therefore, GFP expression decreases.

Why did we use mathematical model?


As mentioned above, it is known that Plac is activated 94-fold under 30 MPa while we didn't know the increase of PL strength under 30 MPa. If PL is activated too much and Plac activity is weaker than PL activity, we can't implement write-function. So, how much is the range of the increase of PL activity under 30 MPa so as to become advantageous to that of Plac? To know this range, we need to use mathematical model.

Classical toggle switch model
Our mathematical model under 0.1 MPa is equal to a classical toggle switch model shown in figure 5-4. nCI is the cooperativity of repression of the lambda promoter, nLacI is the cooperativity of repression of the lac promoter, &alpha;PL is the effective rate of LacI synthesis and &alpha;Plac is the effective rate of CI synthesis. nCI and nLacI are called "Hill coefficient". &alpha;PL and &alpha;Plac depend on strength of promoter-RBS, and are adjustable. Identifying value of nCI and nLacI are required for the modeling. But we know nCI = 3.0 (T. Tian et al., 2006). So, we measured fluorescence intensity various IPTG concentration to identify nLacI.

 Identification of nLacI 

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.

GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weaker by adding IPTG. The characteristics of the lac promoter were calculated by fitting Hill function to the plots shown in figure 5-5, Finally, we obtained nLacI = 2.2.

 Conditions for bistability

We calculated the range of &alpha;PL in which a toggle switch model is bistability. Here, we set &alpha;Plac = 3.0. The result is below.



Pressure model
We proposed pressure-inducible genetic toggle switch model which has additional parameters to the classic toggle switch model. These parameters show the increase of activity by pressure (&beta;(p)Plac or &beta;(p)PL). Under atmospheric pressure (0.1 MPa), &beta;(0.1)Plac = 1.0 and &beta;(0.1)PL = 1.0. On the other hand, under 30 MPa, &beta;(30)Plac = 94 and &beta;(30)PL was not known.

In order to implement write-function, the transit of the system from bistability to monostable (Plac is stronger than PL) by 30 MPa pressure is required. Therefore, we calculated the range of &beta;(30)PL which satisfies the above conditions.

The feasibility of implementation of Write/Erase cycle


According to the result of simulation, we found that we can implement Write/Erase function even if PL is activated 2.5-fold when &alpha;PL = 7.8.

We identified &beta;(30)PL = 1.4 by our wet experiment under 30MPa pressure (figure 5-6). Therefore, we can implement Write/Erase cycle if we choose an appropriate PL - RBS strength.