Coli. TouchImplementation of pressure responsive genetic circuit in E.coli

Ryoji Sekine1, Masashi Kajita2, Yoshihisa Asanuma2, Minori Karibe2, Ryoichi Sato2, Sho Suzuki2, Jun Takimoto2,

Chiaki Kato3, Takako Sato3, Shotaro Ayukawa1, Akio Kobayashi1, Masahiko Uchiyama1, Masayuki Yamamura1, Daisuke Kiga1,4

1Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 2School of Bioscience and Biotechnology, Tokyo Institute of Technology3Japan Agency for Marine-Earth Science and Technology, 4Japan Science and Technology Agency PRESTO

3

We planned to create the touch display with many holes.

Apply pressure

Can’t apply pressure

2. Touch display

1. Pressure inductiontet promoters are known

as pressure responsible onesT. Sato et al., 1995

Ptet-GFP

Tet promoter

(Ptet)RBS GFP Terminator

On pSB6

Constructed

3. Low pressure-inducible promoterB A

Pressure response of Plac

Pressure (MPa)

94 folds

30 MPa is too high to push with a finger

30 MPa = 300 kg/cm2

PCR random mutagenesis

Screening scheme with flow cytometer

4. Write/Erase cycle

PlaccⅠts gfplacI RBS RBS RBS

Pressure (30 MPa)

Heat

Genetic toggle switch

Write-function

Memorize

Erase-function

Memorize

Mathematical model

Pressure model

Introduction

Conclusion

Pressure (30 MPa)

× ?

Under 30 MPa

0.1 MPa

30 MPa

The feasibility of implementation

of Write/Erase cycle

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

Apply 30 MPa pressure to prototype - touch display (two holes)

Acrylicblock cover

Apply pressureProtect

against pressure

Holes8 cm

Plastic tape coverBright Dark

Device for

applying water

pressure

1. Set the display

in pressure vessel

2. Cap the vessel

Prototype - touch

display

4.Cultivate at 37 ℃

3. Apply

pressure to 30

MPa

How to press

0

5

10

15

20

0.1 10 20 30

Flu

ore

scen

ce (

a.u

.)

Pressure (30 MPa)

Clone1

Result of pressure induction

×３

Small molecules

Heat

Light

Pressure

E. coli with GFP expression by pressure

× 94× ?× 94

PLPlac

× 94×?

PL Plac

Step 1

Screening without LacI

Step 2

Screening with LacI

Step 3

Screening with LacI

under low pressure

Mutant library

Low pressure-inducible promoter

finished

Results of sequencing

A

B

mutant

s

Flu

ore

scen

ce

Inte

nsit

y

forward scatter

1234

Mutation Site

123

CAP binding site -35 -10 LacI binding site gfp

It’s only a uniform induction method!!

Advantage of pressure

Non-uniformly Uniformly

Future work

1. Pressure induction: we constructed pressure-inducible genetic circuit and

measured its pressure response.

2.Touch display: we created the prototype - touch display.

3. Low pressure-inducible promoter: We confirmed the feasibility of the

experimental scheme for the development of low pressure inducible promoter.

4. Write/Erase cycle: the feasibility of implementation of the Write/Erase cycle was

confirmed using numerical analysis and measurement of the critical parameter.

0

5

10

15

20

0.1 10 20 30

Flu

ore

sce

nce

(a.u

.)

Pressure (MPa)

Clone1

Clone2３folds

A

B

mutants

1. Pressure induction: Confirm pressure response of the lac promoter.

2. Touch display: Create finished product of touch display.

3. Low pressure-inducible promoter: Screen promoter mutants and LacI mutants.

4. Write/Erase cycle: Construct the genetic circuit

Step 1

Step 2

Step 3

Low High

The increase of PL activity

Numerical analysis to identify upper limit

Where is the upper limit?

Our worksprevious iGEM

projects

Previous study and problem

AcknowledgementsWe thank Hideki Kobayashi, Tadashi Maruyama, Shoji Takeuchi, Tetsuya J. Kobayashi, Yasunori Aizawa, Shigehisa Hirose, Akira Kudo, Ken Kurokawa, Satoshi Nakamura,

Hiroyuki Ohta, Makio Tokunaga, Hiroaki Wachi and Hideya Yuasa for their technical advices. This work is supported by the Art and Crafts Education and Research Support Center

and the School of Bioscience and Biotechnology, Tokyo Institute of Technology.

New input : Pressure