Tokyo University of Agriculture and Technology
Background Spray insecticide Fast- acting!!! Are these Why…?? safe…?? 3
Survey Result of survey Wha hat t ima image ge do do y you ou ha have o e of inse insectic cticide ide? Scar Scary Use Useful ful Poison oisonou ous Af Affec ects ts en envir viron onmen ment ba t badl dly 4
Survey Result of survey Wha hat t ima image ge do do y you ou ha have o e of inse insectic cticide ide? Scar Scary Use Useful ful Poison oisonou ous Af Affec ects ts en envir viron onmen ment ba t badl dly 5
Survey Result of survey Wha hat t ima image ge do do y you ou ha have o e of inse insectic cticide ide? Scary Scar Useful Use ful Poison oisonou ous Affect Af ects s en envir viron onmen ment ba t badl dly 6
Pesticide Insecticide Nervous Nervous ＝ system system Neuron structure Brain Central nerves Peripheral nerves 7
8
Pesticide Insecticide pathway ✔ Metabolic Metabolic ≠ pathway ✔ 9
Insects metabolism Metabolism 【 store 】 【 store 】 Trehalose Glycogen Glycogen Trehalase phosphorylase Glucose Glucose 10
E xterminator coli Concept Trehalase inhibitor production Trehalase inhibitor 11
E xterminator coli Concept Trehalase inhibitor production tr trehalose lose Trehalose production 12
Our strategy Our System Exterminator Coli Outer membrane Trehalase inhibitor production Cytoplasmic membrane Trehalose production
Our strategy Our System Outer membrane Trehalase inhibitor production Cytoplasmic membrane Trehalose production
Trehalose production Glucose-6-phosphate Trehalose-6-phosphate synthetase Trehalose-6-phosphate Trehalose-6-phosphate phosphatase Trehalose
Trehalose production otsA Trehalose-6-phosphate Glucose-6-phosphate synthetase UDP UDP Trehalose-6-phosphate
Trehalose production otsB Trehalose-6-phosphate Trehalose-6-phosphate phosphatase PPi Trehalose
Trehalose production Glucose-6-phosphate otsA Trehalose-6-phosphate synthetase Trehalose-6-phosphate otsB Trehalose-6-phosphate phosphatase Trehalose
Construction + otsB ots RBS RBS otsA otsA RBS RBS S P E X S P E X BBa_K1339010 BBa_K1339011 RBS RBS otsA ots otsB ots RBS RBS Three different S P E X expression levels ! P const constitutive promoters BBa_K1339014 ( P high ) BBa_K1339016 ( P medium ) RBS RBS otsA ots otsB ots RBS RBS S P E X BBa_K1339018 ( P low ) arabinose inducible promoter P BAD BBa_K1339013 ( P BAD ) RBS RBS otsB ots otsA ots RBS RBS S P E X
Evaluation LB medium (600 mM NaCl 2 % Glucose) Cult Cultur ure Measur Measure extr xtrac act
Measurement 1. Expression of 2. Activity of OtsA and OtsB OtsA and OtsB SDS-PAGE TLC (Thin Layer Chromatography) Separate protein by molecular mass 写真 写真 Analyze carbohydrate
OtsA and OtsB expressed under P medium P BAD AD induction (kDa) (kDa) 180 130 180 100 130 75 100 63 OtsA (53.6 kDa) 48 75 63 35 Ots A (53.6 kDa) OtsB (30 kDa) 28 48 17 35 10 Ots B (30 kDa) 28
OtsA and OtsB produced trehalose otsBA under P medium BBa_K1339016 (P medium ) produced trehalose !!! P cons const BBa_K1339018 (P low ) otsA tsA RBS RBS otsB tsB RBS RBS S P E X OtsA OtsB
Trehalose production Glucose-6-phosphate otsA Trehalose-6-phosphate synthetase Trehalose-6-phosphate otsB Trehalose-6-phosphate phosphatase Trehalose
Trehalose production Glucose-6-phosphate otsA Trehalose-6-phophate synthetase Trehalose-6-phophate otsB Trehalose-6-phosphate phosphatase Trehalose
Characterized Existing BioBrick parts otsB RBS BBa_K200017 P mediu medium, lo , low RBS otsB otsB Treh ehalos alose T6P
OtsB expressed under P medium Imperial Nok Nokog ogen en P medium SF P medium SF P medium IF P medium IF SF : soluble fraction P low SF P low SF P low IF P low IF ladder ladder IF : insoluble fraction (kDa) 180 130 100 OtsB expressed 75 63 under P medium !!! 48 35 28 17 10
otsB under P medium produced trehalose otsB Trehalose ehalose T6P
otsB under Pmedium produced trehalose Glucose-6-phosphate otsA Trehalose-6-phophate synthetase Trehalose-6-phophate otsB Trehalose-6-phosphate phosphatase Trehalose
Our strategy Our System Outer membrane Trehalase inhibitor production Cytoplasmic membrane Trehalose production
Trehalase inhibitor Insects metabolism Trehalose Treh ehalas alase inhibitor inh ibitor 3,3’ -diketotrehalose (3,3’dkT) Trehalase Ki = 0.6 mM, IC 50 = 2.5 mM (for trehalase from B. mori ) Glucose K Sode et al ., (2001) FEBS Letters , 489 489, 42-45
Trehalase inhibitor 3,3’ -diketotrehalose Trehalose (3,3’dkT) Ki = 0.6 mM, IC 50 = 2.5 mM (for trehalase from B. mori ) K Sode et al ., (2001) FEBS Letters , 489 489, 42-45
G3DH What is G3DH..? Glucose 3-ketoglucose 1 1 2 2 3 Glucose-3-dehydrogenase 3 (Glucoside-3-dehydrogenase) α γ G3DH β
G3DH What is G3DH..? 3,3’ -diketotrehalose Trehalose Glucose (3,3’dkT) 2’ 2’ 3’ 3’ 1 1’ 1 1’ 2 2 3 3 α γ G3DH β
Constructed Parts for G3DH Construction of bio brick G3DH (from Rhizobium tumefaciens ) RBS RBS a g b BBa_K1339020 S P E X constitutive promoters BBa_K1339022 ( P high ) BBa_K1339023 ( P medium ) RBS RBS a g b BBa_K1339024 ( P low ) SP E X arabinose inducible promoter BBa_K1339021 ( P BAD ) RBS RBS g a b EX SP
Evaluation for G3DH Expression Evaluation 1.SDS-PAGE 2. Activity assay Measure activity by bleaching of dye 写真 写真 Separate proteins by molecular mass
P high , P med , P BAD high , ium , AD (+ (+ ) ) co confir nfirmed med exp xpres ession sion medium G3DH(catalytic subunit): 68 kDa SDS-PAGE P BAD + +
G3DH activity assay Glucose-3-dehydrogenase (G3DH) FAD Ox: Oxidized Red: Reduced Ox Red 3,3’dkT FAD mediator cofactor trehalose Ox Ox Red High absorbance change = High activity
P low has the best G3DH activity 6 5.5 Unit/ml 5 4 D Activity (U/ml) 3 2 1 0 -1 D Activity= activity of each sample – activity of empty vector
P low has the best G3DH activity 6 5 D Activity (U/ml) 4 3 2 1 0 -1
Evaluation of G3DH Our System trehalose 3,3’ -diketotrehalose α G3DH γ β
Evaluation of G3DH Our System trehalose 3,3’ -diketotrehalose α G3DH γ β
We constructed 2 devices Future work Synthesizer for Converter for trehalose trehalase inhibitor production production 43
Future planning : Exterminator Coli Future work Promoter RBS RBS ots otsA ots otsB RBS RBS Trehalose production Promoter a g b RBS RBS Trehalase inhibitor production 44
Future planning : Exterminator Coli Future work 45
For safety Future work 1. To prevent exterminator coli from spreading Lysis 2. To prevent Exterminator coli from affecting other insects Pheromone 46
1. Lysis : BBa_K519020 BBa_K519020 (2011 Tokyo-NoKoGen) 47
1. Lysis 48
2. Pheromone 49
Human Practice 1. School festival at University of Tokyo 2. Survey on synthetic biology 50
1. School festival at University of Tokyo Future work Scho hool ol festival @ Univ @ Univer ersity sity of of T Tok okyo • Introducing the past projects to let people know about synthetic biology and iGEM • Exchanging ideas with iGEMers in Japan 51
2. Survey on synthetic biology Japanese version English version 410 159 52
The ratio of English ver. Japan Indonesia USA Canada Honduras Mexico Azeri Brunei UI Indonesia, Sumbawagen and Minnesota teams cooperated in our survey. 53
Q. What image do you have of "E. coli“ ? Scientist Non scientist Good for science Elegant Cute 5% 7% Positive Positive Positive Negative 40% 56% Negative 37% 55% Negative Infectious Dirty Stinky Scary 54
Q. What do you think about “Exterminator Coli” ? Non-scientist Scientist Interesting Amazing 9% 11% Negative Negative Positive Positive Positive Negative 91% 89% Dirty Infectious Impossible 55
Q. What do you think about “Exterminator Coli” ? 56
Based on the result of survey… 57
Achievement Future work De Device vice Bi BioBri oBrick Wor ork k ✔ 1. Trehalose otsA & 11 production otsB 2. Trehalase ✔ inhibitor G3DH 7 production Gold Gold criteria criteria ✔ 2009 Imperial College Characterization of RBS + otsB an existing BioBrick part (BBa_K200017) 58
Future work Acknowledgement Instructors Prof. Koji Sode, Prof. Kazunori Ikebukuro, Prof. Stefano Ferri Prof. Wakako Tsugawa, Dr. Koichi Abe, Advisors Dr. James Ellinger, Jinhee Lee, Mayumi Nakamura, Ippei Sakamoto Special thanks UI-Indonesia, Sumbawagen, Minnesota, UT-Tokyo 59
Thank you for your attention! 61
Thank you for your attention! Human practice Student dents Maui Nishio, Madoka Nagata, Yuri Ikuta, Nanoha Suzuki, Chika Shono, Saori Fuse, Madoka Okurita, Yasuko Yamagishi, Miki Sanada 62
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