Tokyo University of Agriculture and Technology Background Spray - - PowerPoint PPT Presentation

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Tokyo University of Agriculture and Technology Background Spray - - PowerPoint PPT Presentation

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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

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SLIDE 1
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SLIDE 2

Tokyo University of Agriculture and Technology

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SLIDE 3

Spray insecticide

Fast- acting!!! Why…?? Are these safe…??

Background

3
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SLIDE 4

Result of survey Survey

Wha hat t ima image ge do do y you

  • u ha

have o e of inse insectic cticide ide?

Scar Scary Poison

  • isonou
  • us

Use Useful ful Af Affec ects ts en envir viron

  • nmen

ment ba t badl dly

4
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SLIDE 5

Result of survey Survey

Wha hat t ima image ge do do y you

  • u ha

have o e of inse insectic cticide ide?

Scar Scary Poison

  • isonou
  • us

Use Useful ful Af Affec ects ts en envir viron

  • nmen

ment ba t badl dly

5
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SLIDE 6

Scar Scary Poison

  • isonou
  • us

Use Useful ful

Result of survey Survey

Wha hat t ima image ge do do y you

  • u ha

have o e of inse insectic cticide ide?

6

Af Affect ects s en envir viron

  • nmen

ment ba t badl dly

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SLIDE 7

Nervous system Pesticide Insecticide Nervous system

＝

Brain Neuron structure

Central nerves Peripheral nerves

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SLIDE 8 8
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SLIDE 9

Metabolic pathway Pesticide Insecticide

≠

Metabolic pathway✔

✔

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SLIDE 10

Trehalase Glucose

Insects metabolism Metabolism

Trehalose

【store】

Glucose Glycogen phosphorylase Glycogen

【store】

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SLIDE 11

Concept

Trehalase inhibitor production

Exterminator coli

Trehalase inhibitor

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SLIDE 12

Trehalose production

Concept

Trehalase inhibitor production

Exterminator coli

tr trehalose lose

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SLIDE 13

Our System

Our strategy

Outer membrane Cytoplasmic membrane

Exterminator Coli

Trehalose production Trehalase inhibitor production

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SLIDE 14

Our System

Our strategy

Outer membrane Cytoplasmic membrane

Trehalase inhibitor production Trehalose production

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SLIDE 15

Trehalose production

Glucose-6-phosphate Trehalose-6-phosphate Trehalose

Trehalose-6-phosphate synthetase Trehalose-6-phosphate phosphatase

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SLIDE 16

Trehalose production

Glucose-6-phosphate Trehalose-6-phosphate

UDP UDP

Trehalose-6-phosphate synthetase

  • tsA
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SLIDE 17

Trehalose production

Trehalose-6-phosphate Trehalose

PPi

Trehalose-6-phosphate phosphatase

  • tsB
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SLIDE 18

Trehalose production

Glucose-6-phosphate Trehalose-6-phosphate Trehalose

Trehalose-6-phosphate synthetase Trehalose-6-phosphate phosphatase

  • tsA
  • tsB
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SLIDE 19

Construction

BBa_K1339010 BBa_K1339011 BBa_K1339014 (Phigh) BBa_K1339013 (PBAD)

+

BBa_K1339016 (Pmedium) BBa_K1339018 (Plow)

Three different expression levels !

constitutive promoters

  • ts
  • tsA

RBS RBS

E X S P

  • ts
  • tsB

Pconst

RBS RBS

arabinose inducible promoter

  • ts
  • tsA

RBS RBS

E X S P

  • ts
  • tsB

PBAD

RBS RBS

  • ts
  • tsA

RBS RBS

E X S P

  • ts
  • tsB

RBS RBS

  • tsA
  • tsA

RBS RBS

E X S P

  • ts
  • tsB

RBS RBS

E X S P

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SLIDE 20

Measur Measure extr xtrac act

Cult Cultur ure Evaluation

LB medium (600 mM NaCl 2 % Glucose)

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  • 1. Expression of

OtsA and OtsB

  • 2. Activity of

OtsA and OtsB

Measurement

写真

TLC

(Thin Layer Chromatography) Analyze carbohydrate 写真

SDS-PAGE

Separate protein by molecular mass

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OtsA and OtsB expressed under Pmedium

Ots A (53.6 kDa) Ots B (30 kDa) OtsA (53.6 kDa) OtsB (30 kDa)

PBAD

AD 180 100 130 75 63 48 35 28 17 10

(kDa)

180 100 130 75 63 48 35 28

(kDa) induction

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OtsA and OtsB produced trehalose

  • tsBA under Pmedium

produced trehalose !!!

OtsB OtsA

BBa_K1339016 (Pmedium) BBa_K1339018 (Plow)

  • tsA
tsA RBS RBS E X S P
  • tsB
tsB

Pcons

const RBS RBS
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SLIDE 24

Trehalose production

Glucose-6-phosphate

Trehalose-6-phosphate

Trehalose

Trehalose-6-phosphate synthetase Trehalose-6-phosphate phosphatase

  • tsA
  • tsB
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SLIDE 25

Trehalose production

Glucose-6-phosphate Trehalose-6-phophate Trehalose

Trehalose-6-phophate synthetase

Trehalose-6-phosphate phosphatase

  • tsA
  • tsB
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SLIDE 26

Characterized Existing BioBrick parts

BBa_K200017

  • tsB

RBS

  • tsB

RBS

Pmediu

medium, lo , low

T6P

Treh ehalos alose

  • tsB
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SLIDE 27

OtsB expressed under Pmedium

Pmedium SF Pmedium IF Plow SF Plow IF ladder

SF : soluble fraction IF : insoluble fraction

Pmedium SF Pmedium IF Plow SF Plow IF ladder 180 100 130 75 63 48 35 28 17 10

(kDa)

Imperial Nok Nokog

  • gen

en

OtsB expressed under Pmedium !!!

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SLIDE 28
  • tsB under Pmedium produced trehalose

T6P

Trehalose ehalose

  • tsB
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Glucose-6-phosphate Trehalose-6-phophate Trehalose

Trehalose-6-phophate synthetase

Trehalose-6-phosphate phosphatase

  • tsA
  • tsB
  • tsB under Pmedium produced trehalose
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Trehalose production

Our System

Our strategy

Outer membrane Cytoplasmic membrane

Trehalase inhibitor production

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Trehalose Glucose

Insects metabolism

Trehalase inhibitor

Treh ehalas alase inh inhibitor ibitor

Trehalase

3,3’-diketotrehalose (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

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SLIDE 32

Trehalose 3,3’-diketotrehalose (3,3’dkT)

Trehalase inhibitor

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

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SLIDE 33

What is G3DH..? G3DH γ β

G3DH

α Glucose

1 2 3

3-ketoglucose

1 2 3 Glucose-3-dehydrogenase (Glucoside-3-dehydrogenase)

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SLIDE 34

What is G3DH..? G3DH α γ β

G3DH

Glucose

1 2 3

3,3’-diketotrehalose (3,3’dkT)

1 2 3 1’ 2’ 3’ 1’ 2’ 3’

Trehalose

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SLIDE 35

Construction of bio brick

Constructed Parts for G3DH

BBa_K1339020 b

RBS RBS

E X S P

a g BBa_K1339022 (Phigh) BBa_K1339023 (Pmedium) BBa_K1339024 (Plow)

constitutive promoters

b

RBS RBS

EX SP

a g BBa_K1339021 (PBAD)

arabinose inducible promoter

b

RBS RBS

EX SP

a g

G3DH (from Rhizobium tumefaciens)

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Evaluation

1.SDS-PAGE

写真 写真

  • 2. Activity assay

Evaluation for G3DH Expression

Separate proteins by molecular mass Measure activity by bleaching of dye

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SLIDE 37

Phigh

high,

, Pmed

medium ium,

, PBAD

AD(+

(+)

) co

confir nfirmed med exp xpres ession sion

G3DH(catalytic subunit): 68 kDa

SDS-PAGE

+ +

PBAD

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SLIDE 38

G3DH activity assay

trehalose 3,3’dkT

FAD

Ox Ox Ox Red Red Glucose-3-dehydrogenase (G3DH)

FAD

Ox: Oxidized Red: Reduced

cofactor

mediator

High absorbance change = High activity

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SLIDE 39

DActivity= activity of each sample – activity of empty vector

Plow has the best G3DH activity

5.5 Unit/ml

  • 1

1 2 3 4 5 6 DActivity (U/ml)

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SLIDE 40

Plow has the best G3DH activity

  • 1

1 2 3 4 5 6 DActivity (U/ml)

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SLIDE 41

Our System

Evaluation of G3DH

G3DH α γ β 3,3’-diketotrehalose trehalose

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Our System

Evaluation of G3DH

G3DH α γ β 3,3’-diketotrehalose trehalose

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Future work We constructed 2 devices

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Synthesizer for trehalose production Converter for trehalase inhibitor production

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Future work Future planning : Exterminator Coli

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  • ts
  • tsA

RBS RBS

  • ts
  • tsB
Promoter

RBS RBS

b

RBS RBS

a g

Promoter

Trehalose production Trehalase inhibitor production

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SLIDE 45

Future work Future planning : Exterminator Coli

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SLIDE 46

Future work For safety

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  • 1. To prevent exterminator coli

from spreading

  • 2. To prevent Exterminator coli

from affecting other insects Lysis Pheromone

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SLIDE 47
  • 1. Lysis : BBa_K519020

BBa_K519020 (2011 Tokyo-NoKoGen)

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  • 1. Lysis
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  • 2. Pheromone
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Human Practice

  • 1. School festival at University of Tokyo
  • 2. Survey on synthetic biology
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Future work

  • 1. School festival at University of Tokyo

Scho hool

  • l festival @ Univ

@ Univer ersity sity of

  • f T

Tok

  • kyo
  • Introducing the past projects to let people

know about synthetic biology and iGEM

  • Exchanging ideas with iGEMers in Japan
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SLIDE 52
  • 2. Survey on synthetic biology

159 410

Japanese version English version

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SLIDE 53

The ratio of English ver.

Japan Indonesia USA Canada Honduras Mexico Azeri Brunei

UI Indonesia, Sumbawagen and Minnesota teams

cooperated in our survey.

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  • Q. What image do you have of "E. coli“ ?

Non scientist Scientist 56% 37% 7% 40% 55% 5% Positive Positive Negative Negative Infectious Dirty Stinky Scary Negative Good for science Elegant Cute Positive

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  • Q. What do you think about “Exterminator Coli” ?

89% 11% 91% 9% Scientist Non-scientist Positive Negative Negative Positive Interesting Amazing Positive Dirty Infectious Impossible Negative

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  • Q. What do you think about “Exterminator Coli” ?
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Based on the result of survey…

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Future work

Achievement

De Device vice Bi BioBri

  • Brick

Wor

  • rk

k

  • 1. Trehalose

production

  • tsA &
  • tsB

11

  • 2. Trehalase

inhibitor production G3DH 7 Gold Gold criteria criteria Characterization of an existing BioBrick part

2009 Imperial College RBS + otsB

✔ ✔ ✔

(BBa_K200017)

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SLIDE 59

Future work

Acknowledgement

Advisors

  • Prof. Wakako Tsugawa, Dr. Koichi Abe,
  • Dr. James Ellinger, Jinhee Lee, Mayumi Nakamura,

Ippei Sakamoto

Instructors

  • Prof. Koji Sode, Prof. Kazunori Ikebukuro,
  • Prof. Stefano Ferri

Special thanks

UI-Indonesia, Sumbawagen, Minnesota, UT-Tokyo

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SLIDE 60 61

Thank you for your attention!

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Human practice

Thank you for your attention!

Student dents

Maui Nishio, Madoka Nagata, Yuri Ikuta, Nanoha Suzuki, Chika Shono, Saori Fuse, Madoka Okurita, Yasuko Yamagishi, Miki Sanada

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Nervous system Pesticide insecticide

Neural transmission mechanism acetylcholine signal Acetylcholine degrading enzyme

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Nervous system Pesticide insecticide

Neural transmission mechanism signal

insecticide

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Promoters

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Trehalose Glucose

  • tsA, otsB

Harvest boiling (95 ℃, 5 min) Centrifugation (8,000 g, 15 min, 4℃)

Culture in LB medium (600 mM NaCl, 2 % Glucose), 150 rpm, 37℃, 12 h

Resuspending (Ultra pure water,2 ml/1 g wet cell )

Supernatant

Trehalase reaction (Trehalase 0.007 Unit)

thin-layer chromatography (TLC) Pellet SDS-PAGE Freeze-drying

  • Method. Evaluation of otsBA
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SLIDE 67

Trehalose Trehalose 6- phosphate

  • tsB

Harvest boiling (95 ℃, 5 min) Centrifugation (10,000 g, 10 min, 4℃)

Culture in LB medium (600 mM NaCl, 2 % Glucose)

150 rpm, 37℃, 12 h Resuspending

Supernatant thin-layer chromatography (TLC) Pellet Incubation with Trehalose 6-hosphate Freeze-drying

Sonication

SDS-PAGE

  • Method. Evaluation of otsB
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SLIDE 68

Thin layer chromatography (TLC)

Analyze carbohydrate We used TLC plate coated with silica gel. 1 ml of were spotted

  • nto TLC plates and dried.

TLC plates were developed in acetonitrile– water (7:3)

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SLIDE 69

Standard samples

Glucose Rf value : approximately 0.47 Detection limit : 5 mM Trehalose Rf value : approximately 0.35 Detection limit : 10 mM

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SLIDE 70
  • tsBA OD660 monitoring
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SLIDE 71

Human practice

3,3’dkT

α, , α-Treh ehalose alose (p (ppm) pm) Trehalose lose de deriv river ertiv tive (p (ppm) pm) C-1 93.16 96.56 C-2 70.95 73.61 C-3 72.07 206.56 C-4 69.61 72.50 C-5 72.44 75.28 C-6 60.46 60.46

Table

13C-NMR spectral data of trehalose and trehalose derivative in D2O

K Sode et al., (2001) FEBS Letters, 489 489, 42-45

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SLIDE 72

Human practice

3,3’dkT

TOFMAS analysis → m/Z = 356.1 →→ MW = 360.1 Trehalose MW = 364.1 4 smaller than trehalose

K Sode et al., (2001) FEBS Letters, 489 489, 42-45

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SLIDE 73

Human practice

3,3’dkT

Table Ki and IC50 values of 3,3’dkT of trehalase from pig-kidney and

  • B. mori

Trah ahalase alase Ki (mM mM) IC IC50

50 (mM

mM) pig-kidney 0.2 0.8

  • B. mori

0.6 2.5 * 3,3’dkT did not show any inhibitory effect s on both maltase and mannnosidase.

K Sode et al., (2001) FEBS Letters, 489 489, 42-45

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SLIDE 74

Human practice

Exterminator coli

Tokyo NoKoGen

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SLIDE 75

Trehalose 6-phosphate synthase Trehalose 6-phosphate Trehalose Glucose 6-phosphate Glucose 1-phosphate Glycogen Trehalose 6-phosphate phosphatase Trehalase D-glucose

Insects metabolism

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SLIDE 76

Phigh, Pmedium

Expression of Phigh, Pmedium as inclusion body

  • 1

1 2 3 4 5 6 Dactivity (U/ml)

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SLIDE 77

PBAD

Expression of PBAD(+) as inclusion body

  • 1

1 2 3 4 5 6 Dactivity (U/ml)

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SLIDE 78

pSB 1C3

β γ α

P1 EcoRI-Rv1 EcoRI-Fw1 EcoRI-Rv2 EcoRI-Fw2 P5 P1

α

EcoRI-Rv2

γ β α

P5 EcoRI-Fw1 P1

α γ β α

P5

Construction of G3DH

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SLIDE 79

Our System

Method (G3DH)

Cent Centrifuga rifugation tion (8,000 ,000 g, 15 min, , 15 min, 4℃) Suspending( Suspending(10 10 mM mM SP SPB pH 7.0 1mL ) B pH 7.0 1mL ) Ad Add ar d arabino binose se (OD (OD660

660=0.6)

=0.6)

Pellet SDS-PAGE Measurement of

  • f activity

activity

Resuspending(1 mL/g wet cell) Culture (80 rpm, 37℃, 24 h) Cult Cultur ure e (80 (80 r rpm, 37 pm, 37 ℃, 20 h) , 20 h) (150 rpm, 37℃)

TOP10/pSB1A3-PBAD

  • RBS-G3DH--DT

TOP10/pSB1A3-Phigh, medium, low

  • RBS-G3DH-DT

Harvesting

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SLIDE 80

Protein Expression

DH5a/pTrc G3DHgab (glycerol stock)

3 ml Culture (12 h, 230 rpm, 37 ℃) IPTG induction, OD=1.0 Incubation (30℃, 80 rpm, 20 h), Suspending (10 mM P.P.B. pH 7.0) Sonication (20 sec ON, 20 sec OFF, 5 cycles x 10) Centrifugation (10,000 g, 10 min, 4℃) Ultracentrifugation (104,000 g, 1 h, 4℃) Suspending (1.0 % TritonX-100, 10mM P.P.B. pH 7.0) Ultracentrifugation (104,000 g, 1 h, 4℃) Dialysis (10 mM P.P.B. pH 7.0) Supernatant Pellet

Crude enzyme(soluble membrane)

Supernatant Antibiotic: chloramphenicol 50 μg/ml ampicillin 100 mg/ml Pellet(insoluble fraction) Supernatant(soluble fraction) Pellet(insoluble membrane)

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SLIDE 81

DH5a/pTrcG3DHgab

(kDa) 180 130 100 75 63 48 35 28 17 10

G3DHa 68kDa Activity assay

Activity (U/ml culture) 0.70 Specific activity (U/mg protein) 0.76

0.6 mM PMS 20 ml 0.06 mM DCIP 20 ml 10 mM P.P.B. pH 7.0 120 ml crude enzyme 20 ml 200mM glucose 20 ml

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SLIDE 82

Evaluation of G3DH

0.007 U trehalase SPB

Harvest boiling (95 ℃, 10 min) Centrifugation (8,000 g, 15 min, 4℃)

Culture in LB medium

150 rpm, 37℃, 24 h Suspending (10 mM PSB pH 7.0 , 0.5 ml/1 g cell )

Supernatant

Add Trehalase Measure the trehalase inhibition activity (PMS-DCIP) …compare with empty vector 20 mM Trehalose , 80 rpm, 37 ℃, 10 h Monitoring 0, 5, 8, 20, 30 h, 37℃

DH5a/pTrc G3DHgab/pEC86 (glycerol stock) f.c. 20 mM trehalose SPB

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SLIDE 83

Result of G3DH

0.05 0.1 0.15 0.2 0.25 0.3 10 20 30

activity time (hour)

PMS-DCIP assay

empty G3DH

trehalase inhibition activity

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SLIDE 84

Harvest boiling (95 ℃, 5 min) Centrifugation (8,000 g, 15 min, 4℃)

Culture in LB medium, 20 mM trehalose

150 rpm, 37℃ Suspending (10 mM PSB pH 7.0 , 0.5 ml/1 g cell )

Supernatant

0.007 U Trehalase,SPB

Measure the trehalase inhibition activity (oxidase activity)

20 mM Trehalose , 80 rpm, 37 ℃, 20 h

Evaluation of G3DH

TOP10/PEC86,pSB1A3-PBAD-RBS-G3DH-DT TOP10/PEC86,pSB1A3-empty Pellet f.c. 20 mM trehalose, SPB, 30 h, 37℃ Trehalose 3,3-dKT Trehalose Glucose trehalase

Scheme

Add arabinose (OD660=0.6)

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SLIDE 85

trehalase inhibition activity assay Result of G3DH

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SLIDE 86 87