Team:Tokyo Tech

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Revision as of 03:12, 30 October 2008

Main	Protcol	Parts Submitted to the Registry	Our Team	Acknowledgements

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 ways (small molecule, heat and light) are difficult to induce uniformly. Pressure-input can induce uniformly.

2 Pressure induction

Introduction

A tet promoter is known as a sensitive one to pressure. (T. Sato et al., 1995)

Construction

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

We chose tet promoter(Ptet) as pressure-inducible promoter. We constructed two plasmids - one is Ptet-GFP on pSB6. The other is promoter less-GFP on pSB6 as a negative control.We experimented under 0.1 MPa and 30 MPa pressure.

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 Prototype touch display. figure 3-1 b)

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

Prototype touch display

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

We created a Prototype 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 glasses. 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

Figure 4-2 - PCR random mutagenesis

Figure 4-3 - Screening scheme with flow cytometry

We tried to develop a low pressure inducible promoter by PCR random mutagenesis to lac promoter. Then we screened an E. coli library, with flow cytometry, for promoters that are induced under low pressure. 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.

Step 1 - Fluorescent bacteria without repressor protein were collected by a flow cytometer. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.

Step 2 - This sorted pool contains bacteria bearing both constitutive and pressure-inducible promoter fusions. False positives are removed with repressor protein. All non-fluorescent bacteria are sorted.

Step 3 - A final passage. We sorted bacteria under pressure with repressor protein. Fluorescent bacteria are sorted. False negatives are removed. Therefore bacteria bearing promoter that are low pressure-inducible are enriched.

Results - Sequence and Characterization -

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

Figure 4-4 - Dot plot of cell sorting

Figure 4-5 - Results of sequencing

We sorted fluorescent (A) and non-fluorescent bacteria (B) with a flow cytometer. Then, we analyze these base sequences.

A have mutations in LacI binding site or non-functional DNA.
B have mutations in CAP binding site, -35 or non-functional DNA.

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

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

Mathematical model

Why did we use mathematical model?

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

As mentioned above, it is known that P_lac is activated 94.0-fold under 30 MPa while we didn't know the increase of P_L strength under 30 MPa. If P_L is activated too much and P_lac activity is weaker than P_L activity, we can't implement write-function. So, how much is the range of the increase of P_L activity under 30 MPa so as to become advantageous to that of P_lac? To know this range, we need to use mathematical model.

Classical toggle switch model

figure. 5-4 Classical toggle switch model

figure 5-5. Transfer function of P_lac

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

Identification of n_LacI

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 n_LacI = 2.2.

Conditions for bistability

We calculated the range of α_PL in which a toggle switch model is bistability. Here, we set α_Plac = 3.0. The result is below.

Pressure model

figure 5-6. Pressure-inducible genetic toggle switch 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 (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 was not known.

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

The feasibility of implementation of Write/Erase cycle

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

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

figure 5-8. Pressure-response ability of P_L

We identified β_(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 P_L - RBS strength.

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 === Methods ===
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 Image:Tokyotech2008PCR random mutagenesis.png|Figure 4-2 - PCR random mutagenesis
 Image:Tokyotech2008Screening scheme with flow cytometer.png|Figure 4-3 - Screening scheme with flow cytometry