Team:Tokyo Tech

From 2008.igem.org

(Difference between revisions)

Revision as of 23:54, 28 October 2008

Main	Parts Submitted to the Registry	Our Team	Acknowledgements

Our project

Our project is to create "Coli Touch"!!

What is "Coli touch"?

“Coli Touch” has a pressure sensitive display composed of a E. Coli lawn. When you touch its display, touched section is colored.

Next I'll tell you about “Coli Touch” work system.“Coli Touch” ‘s display has many E. Coli. When you touch this display, pressure input travel to E. Coli in this display. And pressure induce E. Coli expresses GFP.

Why pressure?

“Coli Touch” use pressure as input. Then why we use pressure input? Past input way (small molecule, heat and light) are difficult to induct uniformly. Pressurize can induct unifomly. It's prospect of technological application in confirmatory experiment.

Pressure induction

Construction

figure1. We constructed PtetR-GFP and promoter less-GFP for confirmatory experiment

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

Result ~ activity of Plac ~

figure2.

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

Touch display

Basic touch display

We created a basic touch display made of acrylic glasses.This touch display has two kinds of holes.This device made of Acrylic glasses and has two holes (show figure). 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, 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.

How to press “touch display”

Dilute culture medium by 1% by adding fresh medium and suitable antibiotic (ampicilin; 50㎍/ml). Next, infuse this culture medium into display's holes with oxygen-saturated fluorinert (25% volume of medium). Put this tube into pressure vessel filled with water (1). Next cap the vessel(2). And then aply pressure to the vessel by pressure device(4). Finaly, start incubation at 37℃ immediately at each the vessel for 16h(5).

Result ~ E. coli in the touch display ~

After pressurized the display, After pressurizing, we observed the E. coli in the touch display by a fluorescence microscope.

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

Low pressure-inducible promoter

Figure 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 use as input.

Therefore, we are trying to develop low pressure inducible promoter.

Method

Principle

LacI binds to lacI binding site and repress lac promoter. In addition, pressure of 30 MPa activated the lac promoter. We propose a hypothesis that 30 MPa induce a conformational change in lacI reprssure and change the affinity for lacI binding site weaker. Therefore, lacI can't repress lac promoter under 30 MPa.

Strategy

Figure 1 - RCP random mutagenesis

Figure 2 - Screening system with flow cytometer

We are trying to develop low pressure inducible promoter by PCR random mutagenesis to lac promoter in order to reduce affitity to lacI. And we are screening an E. coli library for promoters that are induced under low pressure using a fluorescence activated cell sorter (FACS). This scheme is based on the ability to separate bacteria with a FACS in response to expression, or lack of expression, of a fluorescent marker.

step 1 - Fluorescent bacteria without repressor protein were collected by FACS. This sorted pool contains bacteria bearing both constitutive and low pressure-inducible promoter.

step 2 - Constitutive promoter are removed with repressor protein and sorting all non-fluorescent bacteria.

step 3 - A final passage through under pressure with repressor protain and sorting for fluorescent bacteria removes false negatives and enriches for bacteria bearing promoter that are low pressure inducible.

Results

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

Sequence and Characterization

Figure 3 - FACS

Figure 4 - Sequensing

We sorted fluorescent (A) and non-fluorescent bacteria (B) with a FACS. 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.

Conclusion

We have successfully demonstrated that it is possible to collect objective promoter by PCR random mutagenesis and screening with a FACS. So we clearly believe that we can screen low pressure inducible lac promoter mutant with this strategy.

Write/Erase cycle

figure** Write/Erase cycle

While we can implement write-function, we also want to implement erase-function and memory-function. Erase-function enable us to erase the painted picture, and memory-function enable us to keep the picture when 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

figure 5-1 genetic circuit model

1. Write-function

30MPa pressure activates P_lac

P_lac expresses CI and GFP

CI represses PL and causes low LacI concentration

P_lac express increasingly CI and GFP ⇒ Bright!!

2. Erase-function

The heat activates P_L

P_L expresses LacI

LacI represses Plac

Therefore, GFP expression decreases

Mathematical model

Why did we use mathematical model ?

figure** above If P_L is not activated or is a bit, write-function is available. bottom If P_L is activated too much, write-function is not unavailable

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, P_lac activity may be weaker than P_L activity and 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 P_lac? To know this range, we used mathematical model.

Classical toggle switch model

figure** Classical toggle switch model

figure 1 Hill function

Our mathematical model under atmospheric pressure is equal to this classical toggle switch model. Where n_CI and n_LacI are hill coefficients, and α_PL and α_Plac are strength of promoter-RBS which are adjustable. We need to identify value of n_CI and n_LacI respectively. But we fortunatly know n_CI = 3.0 (T. Tian et al., 2006). So, we experimented IPTG assay aimed to identify n_LacI.

IPTG assay

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 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 n_LacI = 2.2.

Conditions for bistability

We simulated 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

firuger*** 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, β_(0.1)Plac = 94 and β_(0.1)PL is not known under 30 MPa.

The feasibility of implementation of Write/Erase cycle

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

It is known that Plac activity under 30MPa pressure is 94.0 times stronger than Plac activity under atmospheric pressure, so, β_{30P_lac} = 94.0. If 30MPa additional activity rate of PL (β_{30P_L}) or the ratio of Plac - RBS strength (α_{P_lac}) and PL-RBS strength(α_{P_L}) are not appropriate value, we can't construct PIGTS. So we calculated the domain of appropriate β_{30P_L} and the ratio of α_{P_lac} and α_{P_L}.

figure 5-6 Pressure-response ability of PL

We identified β_{30P_L} = 1.42 by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose P_L - RBS strength range from 2.4 to 7.4.

Our Team

Acknowledgements