Tokyo-NoKoGen Championship Poster

Introduction
Team
Abstract
Insecticides
are not only harmful to insects such as roaches but also pose a risk to mammals. We designed “Exterminator Coli”, an
Escherichia coli machine to exterminate roaches while being completely safe for mammals.
Roaches store their energy in the form of trehalose, which can be converted to glucose by the enzyme trehalase when needed. However,
mammals store their energy in the form of glycogen, which does not require trehalase to be converted to glucose. A trehalase inhibitor
can therefore be used as an insecticide that is harmless for mammals.
In our project, we constructed a precursor production device and an inhibitor production
Exterminator coli
device that can be combined to create Exterminator Coli to produce a safe insecticide.
eats meal
Synthesizer for trehalose
OH
HO
OtsA
We selected 3,3‘-diketotrehalose (3,3’-dkT) as trehalase inhibitor.
3,3’-dkT shows an inhibitory effect toward trehalase because of
structural similarities to trehalose (Sode et al., 2001). To produce
3,3‘-dkT in E. coli, we focused on glucose-3-dehydrogenase (G3DH),
which catalyzes the oxidation of the C-3 hydroxyl group of glucosides,
converting them to the corresponding 3-ketoglucosides (Kojima et
al., 2001). As G3DH has remarkably wide substrate specificity, it can
convert not only monosaccharides but also disaccharides, including
trehalose.
OH
O
OH
O- UDP
OH
HO
UDP-glucose
UDP
HO
OH
O
O
OH OPO 23
O
PPi
OH
OH
OH
OtsB
OH
O
OH
OH
Glucose
OH
Tokyo University of Agriculture & Technology
E-mail: [email protected]
Converter for trehalase inhibitor
To create a device for the production of the trehalase inhibitor, we used the two genes
otsA (trehalose-6-phosphate synthase) and otsB (trehalose-6-phosphate phosphatase),
which are required for trehalose production in E. coli (Eastmond et al., 2003).
OPO32O
Students : Maui NISHIO, Madoka NAGATA, Yuri IKUTA, Yasuko YAMAGISHI, Nanoha
SUZUKI, Chika SHONO Saori FUSE, Madoka OKURITA, Miki SANADA
Advisors : Prof. Wakako TSUGAWA, Dr. Koichi ABE, Jinhee LEE, Mayumi NAKAMURA,
Ippei SAKAMOTO, Dr. James J. ELLINGER
Instructors : Prof. Stefano FERRI,
Prof. Kazunori IKEBUKURO, Prof. Koji SODE
HO
OH
O
OH
OH
OH
HO
O
OH
Trehalose
Trehalose-6-phosphate
Construct
SDS-PAGE analysis
otsBA derived from E. coli
OtsA and OtsB expression were confirmed.
Trehalose
Glucose
Trehalase
Trehalose
2
1 1
3
3,3’-diketotrehalose
2
(3,3’-dkT)
3
3
1 1
2
3
α
G3DH
γ
β
SDS-PAGE Analysis
Construct
G3DH derived from Rhizobium tumefaciens
PConst
BBa_K1339014 (Phigh)
BBa_K1339016 (Pmedium)
BBa_K1339018 (Plow)
OtsA
(53.6 kDa)
OtsB
(30 kDa)
()AA
48
35
28
PBAD
otsBA empty
induction + - + -
PBAD
OtsA
48
35
BBa_K1339022 (Phigh)
BBa_K1339023 (Pmedium)
BBa_K1339024 (Plow)
1. Trehalose production
otsB
Improvement
otsB
Term.
The data was uploaded on the
registry.
Conclusion
We constructed Exterminator Coli, which produces a trehalase inhibitor as a safe
insecticide.
 Synthesizer for trehalose
⇒We confirmed the trehalose
production in our device by using
otsA and otsB genes.
Acknowledgements
 Converter for trehalase inhibitor
⇒We confirmed the trehalase
inhibitor production in our device
by using G3DH
UI-Indonesia, Sumbawagen, Minnesota, UT-Tokyo
Ｃ
48
6
BBa_K200017 (Imperial College(2009))
RBS
68kDa
48
G3DH Activity Assay
DActivity (U/ml)
Migration of Pmedium sample
was the same as that of
standard trehalose sample.
The contruct with Pmedium.
produced trehalose.
68kDa 75
63
BBa_K1339021 (PBAD)
28
RBS
PBAD
G3DH empty
+ - +
G3DH expression was confirmed.
OtsB
Thin layer chromatography (TLC)
75
63
PBAD
Glucose-3-dehydrogenase
(G3DH)
Characterization
of other parts
induction
mw/kDa
100
mw/kDa
100
2. Trehalase inhibitor production
mw/kDa 63
BBa_K1339013 (PBAD)
PConst
z
mw/kDa
63
2
5
5.5 Unit/ml
3,3’dkT
4
3
trehalose
Red
Ox
FAD
cofactor
Ox
mediator
Bleaching
of Dye
Red
2
1
0
-1
DActivity (Unit/ml) =
Activity of each sample –
Activity of empty vector
The scheme of G3DH reaction
The construct with Plow showed
the highest activity.
Ox: Oxidized
Red: Reduced
We investigated G3DH enzymatic
activity by spectrophotometric assay.
Future work considering “Bio Safety”
＋
=
Now we created two devices; trehalose production and trehalase inhibitor
production one. To produce trehalase inhibitor from glucose directly, we are
going to combine these devices and create complete Exterminator Coli.
To reduce risks of Exterminator Coli on the environment…
 Introducing cockroach pheromone expression system
 Introducing the light inducible lysis system
Human Practice
 Questionnaires about synthetic biology in Japanese and English
⇒collected about 500 answers not only from Japan but also from other countries!
 Participation in a school festival
⇒introduced iGEM and synthetic biology to many people!
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