Team:Tokyo Tech

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Parts Submitted to the Registry	Our Team	Acknowledgements

Our project

Our project is to create "Coli Touch"!!

What "Coli touch"?

When you touch this display, touched section colored. How “Coli Touch” work? “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 input. So why we use pressure input? Previous iGEMs, there were various ways as input. For example, small molecules, heat, and light were used. But no one used pressure as input. It’s first reason to use pressure input.

Moreover, 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.

We use pressure sensitive parts. One paper says "One promoter is sensitive to pressure.". So we focused on it.
(cf.Takako Sato, Chiaki Kato,and Koki Horikoshi(1994) Effect of high pressure on gene expression by lac and tac promoters in Escherichia coli.Marine Biotechnology 3:89-92)

Pressure induction

Construction

figure1 We constructed A.PtetR-GFP, B.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([http://partsregistry.org/Part:BBa_K121010 BBa_K121010]), the other is promoter less-GFP on pSB6([http://partsregistry.org/Part:BBa_K121013 BBa_K121013]) as a negative control.

How to press “touch display”

Dilute this culture medium by 1% by adding fresh medium and suitable antibiotic (ampicilin; 50㎍/ml). Next, seal this culture medium with oxygen-saturated fluorinert (25% volume of medium) in polypropylene tubes with parafilm (figure3). Put this tube into pressure vessel filled with water (figure4). Next cup pressure vessel. Finally, pressurize pressure vessel by pressure device(figure5). and then incubation was started at 37℃ immediately at each pressure vessel(original designed) for 16h at 37℃.After cultivation(figure6), the cells were examined by fluorescence microscope.

figure3 Polypropylene tubes with parafilm

figure4 Pressure vessel

|figure5 Pressure device

figure6 Constant-temperature bath

Result

The result was 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

We create devices for confirming pressure response of lac promoter. This is the first step of creating touch display. This device made of Acrylic glasses and has two holes (show figure). Each hole contains tubes and water. Inside tubes E. coli is cultivated. Pressure can travel to inside tubes. One hole (A) is covered with a plastic tape (show figure). Therefore the hole is pressurized.The other (B) is covered with a block made of an acrylic glass. (show figure) Therefore the hole is not pressurized by water.

E.coli type in tubes

* “Ptet” on pSB6 plasmid (E.coli strain; JM109)

After pressurized the display, we observed the E.coli by a fluorescence microscope. The result shows below.

low pressure inducible promoter

It is known that lac promoter is induced under 30 MPa (Kato et al. 1994).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 - Strategy of sorting with FACS

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

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

figure 5-1 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

Differential equation models
figure 5-2 Atmospheric pressure model. It is known that n_{P_L} = 3.0 from earlier studies
figure 5-3 30MPa pressure 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)

figure 5-4 Desirable condition

IPTG assay

figure 1 Hill function

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 n_{P_lac} = 2.15.

Calculate the domain of appropriate parameters

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