Team:Tokyo Tech

From 2008.igem.org

Our project

Our project is to create "Coli Touch"!!

Pressure induction

Construction

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

Result ~ activity of Plac ~

figure2.

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 (figure4). Next cap pressure vessel. Finally, pressurize the vessel by pressure device(figure5). And then start incubation at 37℃ immediately at each the vessel for 16h.

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

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 ?

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_Plac = 2.2.

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_30Plac = 94.0. If 30MPa additional activity rate of PL (β_30PL) or the ratio of Plac - RBS strength (α_Plac) and PL-RBS strength(α_PL) are not appropriate value, we can't construct PIGTS. So we calculated the domain of appropriate β_30PL and the ratio of α_Plac and α_PL.

figure 5-6 Pressure-response ability of PL

We identified β_30PL = 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

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