Team:Tokyo Tech

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Main Protcol Parts Submitted to the Registry Our Team Acknowledgements



Contents

1 Our project

Our project is creation of "Coli Touch"!!

What is "Coli touch"?

Coli Touch” has a pressure sensitive display composed of an E. coli lawn. When you touch its display, touched section is colored.
Next I'll tell you about “Coli Touch” work system. Display of “Coli Touch” has many E. coli. When you touch this display, pressure input travels to E. coli in this display. And pressure applied E. coli expresses GFP.

Why pressure?

Coli Touch” uses pressure as input. Why we use pressure input? Past input way (small molecule, heat and light) are difficult to induce uniformly. Pressurize can induce uniformly.

2 Pressure induction

Introduction

 
It is known tet promoter sensitive to pressure. (T. Sato et al., 1995)

Construction

figure2-1. We constructed Ptet-GFP and promoter less-GFP for confirmatory experiment

 
For confirming pressure-response ability of pressure-inducible promoter, we experimented under 0.1 MPa and 30 MPa pressure. We chose Tet promoter(Ptet) as pressure-inducible promoter.And we constructed two plasmids - one is Ptet-GFP on pSB6. The other is promoter less-GFP on pSB6 as a negative control.

Result ~ activity of Ptet ~

figure2-2. Pressure response of Ptet. Ptet activity increased under 30 MPa

 
The result shows that Ptet activity under 30 MPa pressure is about 3 fold stronger than Ptet activity under 0.1 MPa pressure. Therefore, we confirmed that Ptet was induced under 30 MPa pressure.



          

          

          

          

          

          

          

          

          

          

          

3 Touch display

Touch display (plan)

figure 3-1-b. Design of touch display

 
Touch display that we planned has many holes, and E. coli is in these holes.(figure 3-1 a)
As the first step of creating the touch display,we created basic touch display. figure 3-1 b)

 

 

figure 3-1-a. Plan to create touch display. We created two-holes display as the first step.


Basic touch display

figure 3-2.Basic touch display. The display has two kinds of holes and two kinds of covers.

 

We created a basic touch display made of acrylic glasses. This touch display has two kinds of holes(show figure 3-2). Each hole contains culture medium and E. coli is cultivated in those holes. One hole (A) can be pressurized, because the hole is covered with only a plastic tape.And water pressure conducts into the hole. The other (B) is not pressurized, because the hole is covered with a block made of acrylic glass, water pressure doesn’t conduct into the hole.

 

figure 3-3. Touch display. We pour culture medium into the holes.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

How to apply pressure to “touch display”

 

Dilute culture medium by 1% by addtion of fresh medium and suitable antibiotic (ampicillin; 50㎍/ml). Next, we pour this culture medium into display's holes with oxygen-saturated Fluorinert (25% volume of medium). We put the display into pressure vessel filled with water (1). Next cap the vessel (2). We apply pressure to the vessel by pressure device (4). We start incubation at 37 degrees for 16h (5).


figure 3-4.Protocol for apply pressure to a display.

Result ~ E. coli in the touch display ~

 

After incubation, we observed the E. coli by a fluorescence microscope.

 

 

figure 3-5. Images from fluorescence microscope. E. coli on left image is more bright than right one

 
The touch display successfully regulated GFP expression in E. coli !
                     

4 Low pressure-inducible promoter

Figure 4-1. - Pressure response of Plac

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 tried to develop a low pressure inducible promoter by PCR random mutagenesis to lac promoter. Then we screened an E. coli library for promoters that are induced under low pressure with flow cytometry. This scheme is based on the ability to separate bacteria with a flow cytometer in response to expression, or lack of expression, of a fluorescent marker.

Results - Sequence and Characterization -

We finished step 1. Fluorescent and non-fluorescent bacteria were sorted and we characterized their promoter.

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.

Conclusion

We have successfully demonstrated that it is possible to collect objective promoter by PCR random mutagenesis and screening with a flow cytometry. So we are confident that we can screen low pressure-inducible lac promoter mutant with this methods.

5 Write/Erase cycle

figure5-1. 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

figure 5-2. Genetic toggle switch
  1. Write-function
    1. 30 MPa pressure activates Plac.
    2. Plac expresses CI and GFP.
    3. CI represses PL and decreases LacI expression.
    4. Low LacI expression increases Plac activity. ⇒ Bright!!
  2. Erase-function
    1. The heat activates PL.
    2. PL expresses LacI.
    3. LacI represses Plac.
    4. Therefore, GFP expression decreases.

Mathematical model

Why did we use mathematical model?

figure 5-3. Left If PL is not activated or is a bit, write-function is available. Right If PL is activated too much, write-function is not available

As mentioned above, it is known that Plac is activated 94.0-fold under 30 MPa while we don't know the increase of PL strength under 30 MPa. If PL is activated too much, Plac activity may be weaker than PL activity and 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 Plac? To know this range, we need to use mathematical model.


Classical toggle switch model

Our mathematical model under atmospheric pressure is equal to a classical toggle switch model shown in figure 5-4, where nCI is the cooperativity of repression of the lambda promoter nLacI is the cooperativity of repression of the lac promoter αPL is the effective rate of synthesis of LacI and αPlac is the effective rate of synthesis of CI. nCI and nLacI are called "Hill coefficient". αPL and αPlac depend on strength of promoter-RBS, and are adjustable. strength of promoter-RBS which are adjustable. We need to identify value of nCI and nLacI respectively. But we fortunately 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. We formulated Hill function fitting the data shown in figure 5-5, and the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained nLacI = 2.2.


Conditions for bistability

We simulated the range of αPL in which a toggle switch model is bistability. Here, we set αPlac = 3.0. The result is below.


figure 5-5.PL - RBS strength range from 2.4 to 7.8

Pressure model

figure 5-6. Pressure model

We proposed pressure model which has additional parameters to the classic toggle switch model. These parameters are the increase of activity by pressure (β(p)Plac or β(p)PL). Under atmospheric pressure (0.1 MPa), β(0.1)Plac = 1.0 and β(0.1)PL = 1.0. On the other hand, under 30 MPa, β(30)Plac = 94 and β(30)PL is not known.

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

The feasibility of implementation of Write/Erase cycle

figure 5-7. The domain of appropriate parameters if αPlac = 3.0

According to the result of simulation, we found that we can implement Write/Erase function even if PL is activated 7-folds when αPL = 3.0.

figure 5-8. Pressure-response ability of PL

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

Our Team

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Acknowledgements

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