E. coli - iGEM 2011

Members
TOKYO TECH
Undergrads: Yuko HIGURASHI, Alejandro TSAI, Tomoyuki OHNO, Seohyun KIM, Natsuki KUBO,
Goshi SUGANO, Yuto SUGIUCHI, Takuya TSUBAKI, Maiko HAYASHI, Yumiko HARUHARA,
Hiroki YOSHISE, Tianai LIU, Takashi KITAJIMA
Grad students: Kazuaki AMIKURA, Shotaro AYUKAWA, Ryoji SEKINE, Takefumi MORIYA, Takashi HATA
Instructors: Masayuki YAMAMURA, Akihiko KONAGAYA, Kana ISHIMATSU
Yoh-ichi TAGAWA, Takeharu TSUGE, Daisuke KIGA
Introduction
Cool Prizes
For creating an RPS game between humans and bacteria, we tackled three problems. Firstly, we used two sets of three signaling molecules which
Rain and urea cooler as prizes
correspond each to rock, paper or scissors. Secondly, to know who wins the game, we needed a set of E. coli that act as judges. We constructed the Judge
E. coli by using AND-gate promoters. Finally, we must create randomizers which enable E. coli to choose any of its three signaling molecules with the same
probability in order to be able to play the RPS game fairly and properly. To do so, we designed two randomizers: Conditional Knockout by Recombination and
Survival of one Strain.
Because it was very hot in summer vacation, as a prize for winner in RPS game, we also designed an E. coli that can make it rain and other E. coli that will allow
us to make urea coolers. All this, to make the hot summer more fun and refreshing!
Reference:
[1] O`Dowd, C.D. Aalto, K.Hameri, M.Kulmala, and Thorsten Hoffmann.
Nature, vol. 416, p497-498 (2000)
Choosing one hand
Judge
randomizers which enable E. coli
Using AND gate promoter for Judging
to choose any of three signaling molecules with the same probability
The AND gate promoter can detect two different
signaling molecules as inputs and produce fluorescence
protein as an output to indicate the winner of the game.
We need a set of nine judges because each of the
players has a set of three different signaling molecules.
inducible
lox71
lox2272
lox66
LuxI
lox2272
LasI
LuxS
We designed a Cre-Lox system,
depicted on the left , which allows E.coli
to express one of its three signaling
molecules by conditional knockout.
Regulating reaction time
Each of the 9 Judges contains
one AND gate promoter
lox2272
lox66
lox71
inducible
lox
lox2272
inducible
lox2272
LasI
lox71
LuxS
lox2272
LuxS
To design the randomizer based on Cre-lox recombination,
We constructed one of the Judges
This judge detects human’s paper and E. coli ’s rock, and
shows human’s winning by green fluorescence.
・Plux-lac-GFP(BBa_I751101)[1]
We confirmed the expression of gfp gene is dually
regulated by IPTG and 3OC6-HSL.
we needed two lox cassettes of
different recombination frequency...
[BBa_K649201]
Lo
x
lox2272
Cre
GF
RFPP
Lo
lox2272
-
GFP
Fluorescence Intensity (a.u.)
350
Arginase
Argnine
We succeeded in making
E. coli produce urea.
lox2272
+
GFP x
Lo
GF
lox66
lox71 x RFP P
Cre
JE6852(argR -)
Lo
x
GFP
-
lox
We also performed flux analysis to increase urea production [2].
GFP
+
Our two lox cassettes showed
different recombination frequencies.
By mathmatical modeling, we confirmed that our
artificial urea cycle is already feasible.
310
300
…and we successfully
constructed these
cassettes!
250
200
150
100
2.6
2.6
2.9
Reference: [1] Stefan Schuster, Nat Biotechnol , vol. 18, p326-32 (2000)
[2] Mendel Tuchman, Apple Environ Microbiol, vol. 63, p38-8 (1997)
Human practice
50
0
－
－
＋
＋
3OC6-HSL －
＋
－
＋
IPTG
To create a randomizer, we used three rival
strains each corresponding to Rock, paper, and
scissors. We make them compete for survival
and take the surviving strain to represent the
bacteria’s choice for the RPS game.
we improved two faulty BioBricks:
lsrA promoter and las promoter.
・PlsrA-gfp (BBa_K649104)[2][3][4]
・PlsrA-gfp-PlsrR-LsrR (BBa_K649105)
Our lsrA promoter is repressed by LsrR
35
30
30
25
20
15
9.7
10
We confirmed our
lsrA promoter is
repressed by LsrR.
We created the“iGEM Card Game”
for “Manabi Gate”, an educational event.
Reference: Zorana Carter and Daniela Delneri. Yeast, Vol.185, P.165-171 (2010)
Jean Livet et al. Nature, Vol.450, P.56-63 (2007)
In the process of constructing the Judges,
Fluorescence Intensity (a.u.)
We made use of the fact that
We made Urea
dissolving urea in water is an
cycle in E.coli.
endothermic reaction to make the
Citrulline
urea coolers. We introduced the
rocF gene encoding arginase to
Argininosuccinate make the urea cycle in E. coli [1].
Ornithine
[BBa_K649202]
lux-lac hybrid promoter activity
We fascinated our visitors with the card game! (206 participants!)
We taught Synthetic Biology to them and made a survey.
Do you know the words
"Synthetic Biology"?
Did you become interested
in Synthetic Biology?
②Our New Model
①The Old Model
The rock
strain can’t
survive.
Achievement
Improved
+
・BBa_K649001 PlasI-gfp
5
0
PlsrA-gfp
MG1655
+
PlsrA-gfp
-PlsrR-lsrR
Survivor
・PlasI-gfp (BBa_K649001)[5]
Our lasI promoter is activated by 3OC12-HSL
3500
3300
Fluorescence Intensity (a.u.)
To make it rain, we focused on producing
isoprene. It can act as condensation nuclei
when photooxidized [1]. We currently
conducting the corresponding assays.
3000
2500
2000
1500
1000
500
19
We confirmed our
lasI promoter is
regulated by
3OC12-HSL and LasR.
0
3OC12-HSL
－
＋
Reference
[1] Shotaro Ayukawa et al. BMC Genomics, Vol.11, (2010)
[2] Karina B.Xavier et al. JOURNAL OF BACTERIOLOGY, Vol.187, No.3, p.238-248 (2005)
[3] Liang Wang et al. JOURNAL OF BACTERIOLOGY, Vol.187, No.6, p.2066-2076 (2005)
[4] Ting Xue et al. Cell Research, p.1258-1268 (2009)
[5] Patrick C. Seed et al. JOURNAL OF BACTERIOLOGY, Vol.177, No.3 ,p.654-659 (1995)
AIZAWA Foundation
Durrett & Levin (1996)
(Producer)
(Sensitive)
(Resistant)
Producer
0.32
0.29
0.29
Sensitive
0.29
0.32
0.29
Resistant
0.29
0.29
0.32
+
Tokyo Tech iGEM 2011
In the old model, the producer strain can’t survive, which is a critical limitation as a
randomizer for the RPS game. To be able to create a true randomizer, we modified the
differential equations of the model. We limited the resistance of the colicin-resistant
bacteria. In this case, a type of bacteriocin, produced by colicin-resistant, is toxic to the
colicin-sensitive strain as well as itself. With
our new differential
equations, any of the three types of bacteria can
outcompete the other two strains and ultimately survive by very
small differences between the initial population densities of the strains.
Reference: Durrett and Levin. Journal of Theoretical Biology, vol. 185 ,p165-171(1996)
・BBa_K649104 PlsrA-gfp
・BBa_K649105 PlsrA-gfp-PlsrR-lsrR
Initial population densities (a.u.)
・BBa_K649201 PlacIQ-lox2272-rfp-lox2272-gfp
・BBa_K649202 PlacIQ-lox71-rfp-lox66-gfp
+
・BBa_K649301 Ptrc-rocF
These parts are ALL WORKING PROPERLY!!
Acknowledgement
・Tokyo Institute of Technology
School and Graduate School of Bioscienece Biotechnology
* Department of Life Science Assistant Prof.: Shinya KANEKO
* Department of Bioengineerning Prof.: Yasunori TANJI
* Department of Bioengineerning Assistant Prof.: Kazuaki MIYANAGA
* Bio-Frontier Research Center Prof.:Hiroyuki OHTA
・National Institute for Environmental Studies
* Center for Environmental Measurement and Analysis(Center for Environmental Measurement and Analysis)
/Director :Takashi IMAMURA
* Center for Environmental Measurement and Analysis(Environmental Chemodynamics Section)
/Head :Yoko YOKOUCHI
Collaboration Center for Design
and Manufacturing, Tokyo Tech
Tokyo Tech
130th Anniversary
Foundation