Tokyo-NoKoGen 2015 presents2015.igem.org/files/presentation/Tokyo-NoKoGen.pdfGlycogen phosphorylase Storage Not inhibited!! 13 Engineered E. coli Cellulose Glucose Digestion Trehalose

Tokyo-NoKoGen 2015 presents

A new system to exterminate termites

using Engineered E. coli and pseudo egg

1

Tokyo University of

Agriculture and Technology

Tokyo-NoKoGen

・ 10 Undergraduate members

・ All members belong to

the department of Biotechnology

and Life Science

2

・Introduction

Outline

・ExTermite coli

・Engineered E. coli

・Pseudo egg

・confirm G3DH function

in the termites’ gut extracts

・Biosafety

・Human practice

・Achievement

Kyoko Uchiumi

Kentaro Hiraka

3

Outline

Shunichi Kobayashi

Kinuko Ueno

Kentaro Hiraka

Outline

・Introduction

・Biosafety

・Human practice

・Achievement

Kinuko Ueno

Kyoko Uchiumi

・ExTermite coli


・Pseudo egg



4

Outline

Shunichi Kobayashi

Tumble-down house…

5 5

Introduction

6 6

Introduction

Tumble-down house…

Termite

5 mm http://blog.livedoor.jp/antroom/archives/51515962.html

It was caused by Termites!!

Termites eat woods, papers…

all of the things containing cellulose

7

Introduction

New Extermination System

8

Introduction

・ Easy to use

ExTermite Coli has three features…

Human body

Termite

・ Exterminate termites

・ Harmless to human body

9

Introduction

Our strategy

Our strategy

2. Carry engineered E. coli

by pseudo egg

1. Engineered E. coli

Toxin

3. Carry ExTermite Coli

into termites' bodies

4. Exterminate termites

10

Outline

・Introduction

・Biosafety

・Human practice

・Achievement

Kinuko Ueno

Kyoko Uchiumi

・ExTermite coli


・Pseudo egg

・Confirm G3DH function


11

Outline

Shunichi Kobayashi

Kentaro Hiraka

Our strategy


12

Our strategy

Cellulose Glucose Digestion

Trehalose

Storage

Glucose

Used as

Energy source Trehalase

Engineered E.coli

3,3’ –diketotrehalose

(3,3’-DKT)

Termites

Human

Glycogen Glucose

Energy source Glycogen

phosphorylase Storage

Not inhibited!!

13

Engineered E. coli

Cellulose Glucose Digestion

Trehalose

Storage

Glucose

Used as

Energy source

3,3’ –DKT

Termites

( G3DH )

Glucoside 3-dehydrogenase

Trehalose

14

Engineered E. coli

Trehalase

Engineered E.coli

Inhibit

0

2

4

6

8

10

12

Tre

hala

se a

cti

vit

y (×

10

-2 U

/mL

)

Measurement of inhibitory activity toward trehalase

3,3’-DKT concentration (mM)

Trehalase

3,3’-DKT

inhibits trehalase ! 3,3’-diketotrehalose

(3,3’-DKT)

Measure

Inhibitory Activity of

3,3’-DKT

15

Engineered E. coli

0 0.1 1

Pconstitutive(low)

G3DH α OtsA RBS

Pconstitutive(low)

RBS OtsB

Double terminator T0 terminator

γ β RBS

Evaluation of New biobrick coding OtsA, OtsB, G3DH

Trehalose

3,3’-DKT

UDP-Glucose

Glucose-6 phosphate or

Trehalose-6-

phosphate OtsA OtsB G3DH

16

Engineered E. coli

Production of 3,3’-DKT by new biobrick

Evaluation of our biobrick

UDP-Glucose

Glucose-6 phosphate or

Trehalose-6-

phosphate Trehalose

3,3’-dkT

OtsA OtsB G3DH

SDS-PAGE

63

48

35

28

①Ladder

③G3DH-OtsBA

54 kDa (OtsA)

30 kDa (OtsB)

②empty vector

63 kDa (G3DH)

TLC ①glucose

②trehalose

Trehalose was

produced!

③G3DH-OtsBA

④Negative Control

① ② ③ ① ② ③ ④

17

Engineered E. coli


by pseudo egg

18

Our strategy

Our strategy

・Carry egg and pile them up , and groom it

To distribute engineered E. Coli…

Focused on characteristic of termite

・Groom each other

carry

19

Pseudo egg

Termites recognize

eggs by….

10% (w/v) CaCl2

φ：0.5 mm

E. coli in LB media

&

2% (w/v) Sodium Alginate

Pheromone

( β-glucosidase

and (or)

Lysozyme )

Pseudo Egg

Pheromone β-glucosidase, lysozyme

Size 0.5 mm

0.5 cm

20

Pseudo egg

Pseudo Egg

Count the number of pseudo eggs

transported by termites

0.5 cm

Pseudo egg Formosan subterranean termite

(Coptotermes formosanus)

21

Pseudo egg

Provided by ASANTE Inc.

: Pest extermination company

Pseudo egg

0

20

40

60

80

100

Th

e p

erc

en

tag

e o

f th

e n

um

ber

of

mo

ve

d p

se

ud

o e

gg

Count the number of

moved pseudo egg

Pseudo eggs were

recognized and carried

22

Pseudo egg

Pseudo Egg

Our strategy

1.

2.

3.

4.

The application of the in vivo

experiments using recombinant E. coli is

pending.



23

Our strategy

Outline

Shunichi Kobayashi

・Introduction

・ExTermite coli


・Pseudo egg



・Biosafety ・Human practice

・Achievement

Kinuko Ueno

Kyoko Uchiumi

Kentaro Hiraka

24

Outline

1.

2.

3.

4.


25

Our strategy

Our strategy

about G3DH…

26

G3DH function in termites’ gut

G3DH

FAD

Ox

Red

substrate

(Ox)

substrate

(red)

re-oxidized

G3DH … Glucoside 3-dehydrogenase

G3DH in aerobic condition

O2

H2O

Respiratory chain

e-

Inner membrane

Outer membrane

e-

Periplasmic space

3,3’-diketotrehalose

Trehalose

G3DH

FAD

Reduced

Oxidized

Cytoplasm

Engineered E.coli

e-

G3DH in anaerobic condition

O2

Anaerobic

Termites’ gut

Less O2

Respiratory chain Inner membrane

Outer membrane

Oxidized

Periplasmic space

Trehalose

G3DH

FAD

Reduced

3,3’-

diketotrehalose

Cytoplasm

e-

G3DH in aerobic condition

O2

Electron acceptor

Oxidized

Reduced

Anaerobic

In termites’ gut

Less O2

Respiratory chain Inner membrane

Outer membrane

Trehalose

G3DH

FAD

Reduced

Oxidized

3,3’-diketotrehalose

Periplasmic space

Cytoplasm

e-

WE CE RE G3DH

FAD

Ox

Red

Evaluation of G3DH function in termites’ gut extracts

Get gut extracts from termites

gut extracts

oxidized

reduced

e-

Detect response current using electrodes

Termites

Crush!!

3,3’-DKT

Trehalose

Electrodes

33


0

0.3

0.6

0.9

1.2

Curr

ent

(μA

)

G3DH + Trehalose

+ - Termites’

gut extracts

Evaluation of G3DH using termites’ gut extracts with electrodes

FAD

Ox

Red

trehalose

3,3’-dkT

Intestinal guts

oxidized

reduced

e-

G3DH can work

in termites’ gut extracts

difference of current!!

Scheme of electron transfer


Discussion about electron acceptor

Lignin

degradation

Quinones Electron

acceptor?

35


G3DH

FAD

Ox

Red

Confirmation of G3DH function in termites’ gut solution

oxidized

reduced

e-

Detect the response current using electrodes

WE CE RE

Crush!!

3,3’-DKT

Trehalose

Electrodes

Quinones…? Our Extermite coli can

work in termites’ gut !!

36


For summary…

37

For summary

For summary…

G3DH,OtsA, OtsB

were expressed

from our NEW biobrick!

Inhibitory activity

was confirmed!


38

For summary

For summary…

Making pseudo eggs

Pseudo eggs were

recognized and carried by termites


by pseudo egg

39

For summary

For summary…

G3DH functioned

in termites’ gut extracts!




Pending

40

For summary

Future work…

41

Future work

For practical use,

ingest Engineered E. coli into termites.

・Introduction

・Biosafety

・Human practice

・Achievement

Kinuko Ueno

Kyoko Uchiumi

Kentaro Hiraka

Shunichi Kobayashi

・ExTermite coli


・Pseudo egg



42

Outline

Outline

X X

X X

Destruction of the ecosystem !

Most of insects have the same metabolic pathway

43

Biosafety

Think about biosafety…

44

Biosafety

Prevent the Destruction of the ecosystem

Focus on habit of termites

Dark place Outside = Exposed to light

Spread to…

Using light inducible system

How to prevent the destruction of ecosystem ?

Biosafety

PCB

HK

CcaS

CcaR

CcaS/CcaR two component regulation system

target gene

PcpcG2

Colicin E1

・Forming a pore in

cytoplasmic membrane ・Depolarizing the membrane

RBS T0

Activate!

Bind!

Transcription !

PCB

HK

CcaR

PCB Haem

PCB production system

BV

colicin E1

Light inducible cell killing system

46

Dark condition Light irradiated condition

DH5α / introduced the light inducible cell killing system

Grew up well Not grew up

too much

Confirm the function !!

Biosafety

Function evaluation

Devised and designed

the light inducible cell killing

system for biosafety !

Constructed New Biobrick !

Evaluate the function !

PcpcG2

colicin E1

RBS T0

47

Biosafety

Light induced cell killing system

・Introduction

・Biosafety

・Human practice

・Achievement

Kinuko Ueno

Kyoko Uchiumi

Kentaro Hiraka

・ExTermite coli


・Pseudo egg



48

Outline

Outline

Shunichi Kobayashi

Human practice

“The May Festival” school festival in the University of Tokyo

High school in Tokyo

( Joshi-Gakuin, private girls school )

Survey with UI-Indonesia

・Short lecture

・Questionnaire

・Questionnaire

・Presentation

・Discussion

・Questionnaire

http://www.joshigakuin.ed.jp/schoollife/facilities.html

49

Human practice

Question. What impressions of E. coli do you have?

After our

presentation

In Japan

50

Human practice

Good image

- Available

- Safe

- Interesting

…etc

Bad image

- Dirty

- Dangerous

- Not interesting

…etc

Human practice

Question. What impressions of E. coli do you have?

After our

presentation

In Japan

51

Human practice

Good impression

Bad impression

Good image

- Available

- Safe

- Interesting

…etc

Bad image

- Dirty

- Dangerous

- Not interesting

…etc

The impression of E. coli

was improved !!

Bad impression

Good

impression

Human practice

Question. What images of E. coli do you have?

After our

presentation

In Indonesia

52

Bad impression

Good impression

Good image

- Available

- Safe

- Interesting

…etc

Bad image

- Dirty

- Dangerous

- Not interesting

…etc

The impression of E. coli

was not changed…

Human practice

Human practice

little opportunity for Japanese people to know about synthetic biology

We have to broaden the knowledge of synthetic biology

53

Human practice

After our

presentation

In Japan

Human practice

Question. How do you want to use E. coli in your lifestyle?

In Indonesia In Japan

extermination

of harmful insects

Extermination system

using E. coli is needed!!

54

Human practice

Human practice

■Extermination of

harmful insects

Removal of the fungi at your

bathroom

Small bioenergy as battery

Perc

en

tag

e o

f re

sp

on

de

nt

22%

18% 17%

Question. How do you want to use E. coli in your lifestyle?

Answers from high school

55

Human practice

Human practice

Collaboration

Tokyo-NoKoGen 2015 collaborated with….

Participate in May Festival with… ・UT- Tokyo ・Nagahama ・Tokyo_Tech ・KAIT_Japan

Exchange and translate questionnaire ・UI-Indonesia team

http://2015.igem.org/Team:Tokyo-NoKoGen/Practices

56

Collaboration

☑ Collaborate with other teams

☑ Evaluate the function of previous Biobrick

・Characterize G3DH (BBa_K1339005)

・UT- Tokyo ・Nagahama ・Tokyo_Tech ・KAIT_Japan ・UI-indonesia

☑ Expand Human Practices activity

57

Achievement

Attribution

Tokyo-NoKoGen 2015

10 students, 4 advisors, and 2 instructors

Team members

:All of the works were done

only by team members

Our advisors!!

: Teach us experiments

: Give us some advice

: Support our project

Instructors

: Support our project

: Supervise our activities

58

http://2015.igem.org/Team:Tokyo-NoKoGen/ACK

Attribution

Instructors

Advisors

Special thanks

Dr. Koji Sode, Dr. Kazunori Ikebukuro

Jinhee Lee, Maui Nishio, Madoka Nagata

Acknowledgement

59

Dr. Stefano Ferri (Shizuoka University)

Dr. Wakako Tsugawa

Mr. Shinya Sasaki (ASANTE)

UI-Indonesia, UT-Tokyo, Nagahama

Acknowledgement

Thank you for your attention!

60

Presented by

Tokyo-NoKoGen 2015

5 seconds

Introduction

ExTermite Coli is targeting…

Formosan subterranean termite

Japanese damp-wood

termite

Japanese subterranean termite

We use these termites

in our experiments !!

63

Production of G3DH

Trehalose

3,3’ –dkt

G3DH

Engineered E. coli

Activity assay of G3DH

G3DH-OtsBA Empty vector

Substrate：trehalose

64

Production of G3DH

Engineered E. coli

Activity assay of G3DH

65

Halomonas sp. α-15

gram-negative, rod-shaped marine bacterium isolated from seawater

Glucose 3-dehydrogenase

(G3DH)

monomeric enzyme with 63 kDa

oxidizes 3rd hydroxy group of pyranose

broad substrate specificity

M9Sα plate medium

12 h cultivate

Purification of G3DH from Halomonas sp. α-15

100 mL YM9Sα medium

12 h cultivate 30℃, 120 rpm

Halomonas sp. α-15

3.0 mL M9Sα medium

2L YM9S lac0.8 % medium

30 h cultivate 30℃, 600 rpm

Culture solution

Harvest and Wash (0.85 % NaCl)

5000 g, 4℃, 10 min

20 mM Potassium phosphate buffer (P.P.B) pH 7.0

Wet cells

French press

10,000 g, 20 min, 4 ℃

Supernatant

106,000 g, 60 min, 4 ℃

Soluble fraction

Anion exchange chromatography

Dialyze

Hydrophobic interaction chromatography

Dialyze

Purified fraction reaction with

trehalose 3,3’-diketotrehalose

concentration

Anion exchange chromatography

3 4 5 6 7 8

Confirmation of activity

Hydrophobic interaction chromatography

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21

Confirmation of activity

substrate

glucose 10 mM PMS/DCIP

α-methyl-D-glycoside 100 mM

PMS/DCIP U/mg U/mg

fraction no.

1,2 9.7 7.3 3,4 12 9 5,6 11 9.6

7,8,9 6.9 5.7

66 kDa

Cr F 3 4 5 6 7 Co F 1,2 3,4 5,6 7,8,9 16 17

Cr: crude fraction F: flowthrough Co: concentration 3-7

74

Evaluation of function

Supplement

ccaR

PcpcG2

Colicin E1

RBS

T0

ccaS

AmpR

ori

pSB1A3-CcaSR-

PcpcG2-ColicinE1

pcyA

ho1

Pconst

CmR

ori

pSB4C5-PCB

Light inducible cell killing system PCB production system

E. coli Transformed using above two plasmids

Documents

Tokyo-NoKoGen 2015 presents2015.igem.org/files/presentation/Tokyo-NoKoGen.pdfGlycogen phosphorylase Storage Not inhibited!! 13 Engineered E. coli Cellulose Glucose Digestion Trehalose