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
