tokyo university of2014.igem.org/files/presentation/tokyo-nokogen_championship.pdf · know about...

Tokyo University of

Agriculture and Technology

Spray

insecticide

Fast-

acting!!!

Why…??

Are these

safe…??

Background

3

Result of survey Survey

What image do you have of insecticide?

Scary

Poisonous

Useful

Affects

environment badly

4



Scary

Poisonous

Useful

Affects

environment badly

5

Scary

Poisonous

Useful



6

Affects

environment badly

Nervous

system

PesticideInsecticide

Nervous

system＝

Brain

Neuron structure

Central nerves

Peripheral nerves7

Metabolic

pathway

PesticideInsecticide

≠ Metabolic

pathway✔

✔9

Trehalase

Glucose

Insects metabolismMetabolism

Trehalose

【store】

Glucose

Glycogen

phosphorylase

Glycogen

【store】

10

Concept

Trehalase inhibitor production

Exterminator coli

Trehalase inhibitor

11

Trehalose

production

Concept


Exterminator coli

trehalose

12

Our SystemOur strategy

Outer

membrane

Cytoplasmic

membrane

Exterminator Coli

Trehalose production



Outer

membrane

Cytoplasmic

membrane




Glucose-6-phosphate

Trehalose-6-phosphate

Trehalose


synthetase


phosphatase


Glucose-6-phosphate


UDP

UDP


synthetase

otsA



Trehalose

PPi


phosphatase

otsB


Glucose-6-phosphate


Trehalose


synthetase


phosphatase

otsA

otsB

Construction

BBa_K1339010 BBa_K1339011

BBa_K1339014 (Phigh

)

BBa_K1339013 (PBAD

)

+

BBa_K1339016 (Pmedium

)

BBa_K1339018 (Plow

)

Three different

expression levels !

constitutive promoters

otsARBS

E X S P

otsB

Pconst

RBS

arabinose inducible promoter

otsARBS

E X S P

otsB

PBAD

RBS

otsARBS

E X S P

otsB RBS

otsARBS

E X S P

otsBRBS

E X S P

Measure extract

Culture

Evaluation

LB medium

(600 mM NaCl

2 % Glucose)

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

OtsA and OtsB expressed under Pmedium

Ots A (53.6 kDa)

Ots B (30 kDa)

OtsA (53.6 kDa)

OtsB (30 kDa)

PBAD

180

100

130

75

63

48

35

28

17

10

(kDa)

180

100

130

75

63

48

35

28

(kDa)induction

OtsA and OtsB produced trehalose

otsBA under Pmedium

produced trehalose !!!

OtsB

OtsA


)

BBa_K1339018 (Plow

)

otsARBS

E X S P

otsB

Pconst

RBS


Glucose-6-phosphate


Trehalose


synthetase


phosphatase

otsA

otsB


Glucose-6-phosphate

Trehalose-6-phophate

Trehalose


synthetase


phosphatase

otsA

otsB

Characterized Existing BioBrick parts

BBa_K200017

otsBRBS

otsBRBS

Pmedium, low

T6PTrehalose

otsB

OtsB expressed under Pmedium

Pm

ed

iu

mS

F

Pm

ed

iu

mIF

Plow

SF

Plow

IF

la

dd

er SF : soluble fraction

IF : insoluble fraction

Pm

ed

iu

mS

F

Pm

ed

iu

mIF

Plow

SF

Plow

IF

la

dd

er

180

100

130

75

63

48

35

28

17

10

(kDa)

Imperial Nokogen

OtsB expressed

under Pmedium

!!!

otsB under Pmedium

produced trehalose

T6PTrehalose

otsB

Glucose-6-phosphate


Trehalose


synthetase


phosphatase

otsA

otsB

otsB under Pmedium produced trehalose



Outer

membrane

Cytoplasmic

membrane


Trehalose

Glucose

Insects metabolismTrehalase inhibitor

Trehalase

inhibitor

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, 42-45

Trehalose


(3,3’dkT)

Trehalase inhibitor

Ki = 0.6 mM,

IC 50

= 2.5 mM

(for trehalase from B. mori)


What is G3DH..?

G3DHγ

β

G3DH

α

Glucose

1

2

3

3-ketoglucose

1

2

3Glucose-3-dehydrogenase

(Glucoside-3-dehydrogenase)

What is G3DH..?

G3DHα

γ

β

G3DH

Glucose

1

2

3


(3,3’dkT)

1

2

3

1’

2’

3’

1’

2’

3’

Trehalose

Construction of bio brickConstructed Parts for G3DH

BBa_K1339020bRBS

E X S P

a g

BBa_K1339022 (Phigh

)


)

BBa_K1339024 (Plow

)

constitutive promoters

bRBS

EX SP

a g

BBa_K1339021 (PBAD

)

arabinose inducible promoter

bRBS

EX SP

a g

G3DH (from Rhizobium tumefaciens)

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

Phigh

, Pmedium

, PBAD

(+) confirmed expression

G3DH(catalytic subunit): 68 kDaSDS-PAGE

+ +

PBAD

G3DH activity assay

trehalose

3,3’dkT

FAD

Ox

Ox Red

Red

Glucose-3-dehydrogenase

(G3DH)FAD

Ox: Oxidized

Red: Reduced

cofactor

mediator

High absorbance change = High activity

DActivity=

activity of each sample – activity of empty vector

Plow

has the best G3DH activity

5.5 Unit/ml

-1

0

1

2

3

4

5

6

DA

cti

vit

y(U

/m

l)

Plow

has the best G3DH activity

-1

0

1

2

3

4

5

6

DA

cti

vit

y(U

/m

l)

Our SystemEvaluation of G3DH

G3DHα γ

β

3,3’-diketotrehalosetrehalose

Future workWe constructed 2 devices

43

Synthesizer for

trehalose

production

Converter for

trehalase inhibitor

production

Future workFuture planning : Exterminator Coli

44

otsARBS otsB

Promoter

RBS

bRBS a g

Promoter



Future workFuture planning : Exterminator Coli

45

Future workFor safety

46

1. To prevent exterminator coli

from spreading

2. To prevent Exterminator coli

from affecting other insects

Lysis

Pheromone

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

Future work1. School festival at University of Tokyo

School festival @ University of Tokyo

• 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

159410

Japanese version English version

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“ ?

Non scientistScientist

56%

37%

7%

40%

55%

5%

Positive Positive

Negative

Negative

Infectious

Dirty

Stinky

Scary

Negative

Good for science

Elegant

Cute

Positive

54

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

89%

11%

91%

9%

Scientist

Non-scientist

Positive

NegativeNegative

Positive

Interesting

Amazing

Positive

Dirty

Infectious

Impossible

Negative

55

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

56

Based on the result of survey…

57

Future workAchievement

Device BioBrick Work

1. Trehalose

production

otsA &

otsB11

2. Trehalase

inhibitor

production

G3DH 7

Gold criteria

Characterization of

an existing BioBrick part

2009 Imperial College

RBS + otsB

✔

✔

✔(BBa_K200017)

58

Future workAcknowledgement

AdvisorsProf. 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

59

61

Thank you for your attention!

Human practiceThank you for your attention!

Students Maui Nishio, Madoka Nagata, Yuri Ikuta,

Nanoha Suzuki, Chika Shono, Saori Fuse, Madoka Okurita,

Yasuko Yamagishi, Miki Sanada 62

Nervous

system

Pesticideinsecticide

Neural transmission mechanism

acetylcholine

signal

Acetylcholine

degrading

enzyme

64

Nervous

system

Pesticideinsecticide

Neural transmission mechanism

signal

insecticide

65

Promoters

TrehaloseGlucoseotsA, 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

TrehaloseTrehalose 6-phosphate

otsB

Harvest



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

Thin layer chromatography (TLC)

Analyze carbohydrate

We used TLC plate coated with

silica gel. 1 ml of were spotted

onto TLC plates and dried.

TLC plates were developed in acetonitrile–

water (7:3)

Standard samples

Glucose

Rf value : approximately 0.47

Detection limit : 5 mM

Trehalose

Rf value : approximately 0.35

Detection limit : 10 mM

otsBA OD660 monitoring

Human practice3,3’dkT

α, α-Trehalose (ppm) Trehalose derivertive (ppm)

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 D

2O



TOFMAS analysis

→ m/Z = 356.1

→→ MW

= 360.1

Trehalose

MW

= 364.1

4 smaller than

trehalose



Table

Kiand IC

50values of 3,3’dkT of trehalase from pig-kidney and

B. mori

Trahalase Ki(mM) IC

50(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.


Human practiceExterminator coli Tokyo NoKoGen

75

Trehalose 6-phosphate synthase

Trehalose 6-phosphate

Trehalose

Glucose 6-phosphate

Glucose 1-phosphate

Glycogen

Trehalose 6-phosphatephosphatase

Trehalase

D-glucose

Insects metabolism

76

Phigh, Pmedium

Expression of Phigh, Pmedium

as inclusion body

-1

0

1

2

3

4

5

6

Da

cti

vit

y(U

/m

l)

PBAD

Expression of PBAD(+)

as inclusion body

-1

0

1

2

3

4

5

6

Da

cti

vit

y(U

/m

l)

pSB 1C3

βγα

P1

EcoRI-Rv1

EcoRI-Fw1

EcoRI-Rv2

EcoRI-Fw2

P5

P1

α

EcoRI-Rv2

γ βα

P5

EcoRI-Fw1

P1

α γ βαP5

Construction of G3DH

Our SystemMethod (G3DH)

Centrifugation

(8,000 g, 15 min, 4℃)

Suspending(10 mM SPB pH 7.0 1mL )

Add arabinose

(OD660

=0.6)

Pellet

SDS-PAGE Measurement of activity

Resuspending(1 mL/g wet cell)

Culture

(80 rpm, 37℃, 24 h)

Culture

(80 rpm, 37 ℃, 20 h)

(150 rpm, 37℃)

TOP10/pSB1A3-PBAD

-RBS-G3DH--DT

TOP10/pSB1A3-Phigh, medium, low

-RBS-G3DH-DT

Harvesting

Protein ExpressionDH5a/pTrc G3DHgab (glycerol stock)

3 ml Culture (12 h, 230 rpm, 37 ℃)

IPTG induction, OD=1.0Incubation (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)


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/mlampicillin 100 mg/ml

Pellet(insoluble fraction)

Supernatant(soluble fraction)

Pellet(insoluble membrane)

DH5a/pTrcG3DHgab

(kDa)

180

130100

7563

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

Evaluation of G3DH

0.007 U trehalaseSPB

Harvest



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 trehaloseSPB

Result of G3DH

0

0.05

0.1

0.15

0.2

0.25

0.3

0 10 20 30

acti

vity

time (hour)

PMS-DCIP assay

empty

G3DH

trehalase inhibition activity

Harvest



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 Glucosetrehalase

Scheme Add arabinose (OD660=0.6)

trehalase inhibition activity assayResult of G3DH

tokyo university of2014.igem.org/files/presentation/tokyo-nokogen_championship.pdf · know about...

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