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3

4

5

6

Vitamin Synthesis

Biofuel Drug

Thermo-induced Device

7

RBS GFP J23114

RBS CI

PcI

J23101 37 oC RBS

GFP

37oC induced Device

42oC induced Device

J23114 RBS CI

PcI

RBS GFP

42oC induced Device

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42oC Induced Device

42oC Induced Device - Modeling

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GFPtgGFPdt

GFPdGFPTemp

J23114 RBS CI

PcI

RBS GFP

0%

20%

40%

60%

80%

100%

25℃ 37℃ 42℃

Relative Protein Expression Activity

J23101 37 oC RBS

GFP

0

500

1000

1500

2000

2500

3000

3500

4000

0 2 4 6 8 10

MEFL

HR

27 ℃

32 ℃

37 ℃

42 ℃

10

37oC Induced RBS

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0%

20%

40%

60%

80%

100%

120%

25℃ 30℃ 37℃ 42℃

Relative Protein Expression Activity

The relative translational activity of thermo-induced RBS BBa_K115002 at 25°C, 30°C, 37 °C and 42°C were estimated.

37oC Induced RBS - Modeling

J23101 37oC RBS

GFP

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Farnesyl diphosphate

Phytoene

Lycopene

β-carotene

Geranylgerabyl diphosphate

crtE

crtB

crtI

crtY

crtZ

30oC

37oC device

42oC device

Carotenoid Synthesis Pathway

42oC induced Device

crtZ

J23114 RBS CI

PcI

RBS

crtY 370 RBS

J23103

crtE crtI crtB RBS RBS RBS

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Carotenoid Biobrick

30 oC

14

crtY 370 RBS

J23103

crtE crtI crtB RBS RBS RBS

42oC induced Device

crtZ J23114

RBS CI

PcI

RBS

30oC

Lycopene

30 oC

15

crtY 370 RBS

J23103

crtE crtI crtB RBS RBS RBS

42oC induced Device

crtZ J23114

RBS CI

PcI

RBS

crtY 370 RBS

37oC

30oC 37oC

β-carotene

30 oC

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crtY 370 RBS

J23103

crtE crtI crtB RBS RBS RBS

37oC

J23114 RBS CI

PcI

RBS crtZ crtZ J23114

RBS CI

PcI

RBS

42 oC

Zeaxanthin

30oC 37oC 42oC

30oC 37oC 42 oC

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Results

30oC

37oC

42oC

We can make the pathway stop at the certain step by controlling temperature.

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Vitamin Synthesis Biofuel

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Biofuel

Motivative

Gene strains Length(bp)

ilvC ketol-acid reductoisomerase,

NAD(P)-binding

Escherichia coli str. K-12

substr. MG1655

1473

ilvD dihydroxyacid dehydratase,

Escherichia coli str. K-12

substr. MG1655

1848

alsS

alpha-acetolactate synthase

protein,

Bacillus subtilis 1713

kivd

alpha-ketoisovalerate

decarboxylase,

Lactococcus lactis subsp.

lactis KF147

1734

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Clone genes

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Modify the RE Site P E X S E P P X

Mutate 4 RE site

alsS

E X S P E E

Mutate 2 RE site

ilvC

E X S P P

Mutate 1 RE site

ilvD

P E X S E

Mutate 1 RE site

kivd

alsS

ilvC

ilvD

kivd

Pyruvate

2-Ketoisovalerate

Isobutyraldehyde

Isobutanol

2-Acetolactate

Glucose

2,3-Dihydroxy-isovalerate

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Butanol Synthesis Pathway

Plac

RBS alsS ilvD ilvC RBS RBS RBS kivd

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alsS

ilvC

ilvD

kivd

Pyruvate

2-Ketoisovalerate

Isobutyraldehyde

Isobutanol

2-Acetolactate

Glucose

2,3-Dihydroxy-isovalerate

In Traditional Circuit

ADH

Pyruvate

2-Ketoisovalerate

Isobutyraldehyde

2-Acetolactate

Glucose

2,3-Dihydroxy-isovalerate

alsS

ilvC

ilvD

kivd

37 oC

30 oC

24 Isobutanol

Butanol Synthesis Pathway

Plac

RBS alsS ilvD ilvC tetR RBS RBS 370 RBS

RBS kivd

Ptet

Pyruvate

2-Ketoisovalerate

Isobutyraldehyde

Isobutanol

2-Acetolactate alsS

ilvC

ilvD

kivd

Glucose

2,3-Dihydroxy-isovalerate

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Butanol Biobrick

ADH

2-Ketoisovalerate

kivD

Ptet

RBS

Pyruvate

2-Acetolactate alsS

ilvC

ilvD

Isobutyraldehyde

Isobutanol

kivd

Glucose

2,3-Dihydroxy-isovalerate

37 oC

kivd

Ptet

RBS

tetR

2-Ketoisovalerate

Plac

RBS alsS ilvD ilvC tetR RBS RBS 370 RBS

37oC

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ADH

kivd

Ptet

RBS

Pyruvate

2-Ketoisovalerate

2-Acetolactate alsS

ilvC

ilvD

Isobutyraldehyde

Isobutanol

kivd

Glucose

2,3-Dihydroxy-isovalerate

30 oC

30 oC

kivd 2-Ketoisovalerate

Isobutanol

Plac

RBS alsS ilvD ilvC tetR RBS RBS 370 RBS

tetR 370 RBS

37oC 30oC

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0hr.(Without 30oC incubation) 24hr.(Incubation at 30oC)

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Isobutanol Produce Test in 30oC

30⁰C 42⁰C

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Isobutanol Produce Test in 30oC, 42oC

0

50

100

150

200

250

300

350

400

450

30 ℃ 37 ℃ 42 ℃

Iso

bu

tan

ol c

oc.

(p

pm

)

Temperature Non-temperature controlled device

Low-temperature released device

Plac

RBS alsS ilvD ilvC tetR RBS RBS 370 RBS

RBS kivd

Ptet

Plac

alsS ilvD ilvC RBS kivd RBS RBS RBS

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Comparison of Isobutanol Production

30oC 37oC 42oC

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Biofuel Drug

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IPA imine

IPA imine dimer

I-tryptophan

Protodeoxyviolaceinic acid(PVA)

Deoxychromoviridans Protoviolaceinic acid

Violacein

Biosynthesis Pathway

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Protoviolaceinic acid

I-tryptophan

Protodeoxyviolaceinic acid(PVA)

IPA imine

vioA 30⁰C

37⁰C

42⁰C

IPA imine dimer

Deoxychromoviridans

Violacein

vioB

vioE

vioD

vioC

vioC 42⁰C

Violancein Pathway

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Ptet

RBS vioA vioB vioE RBS RBS

tetR

Plac

vioD 370 RBS

370 RBS

A.

B.

C.

Violancin Biobrick

vioC RBS lacI RBS tetR

42oC induced Device

J23114

RBS CI

PcI

RBS

I-tryptophan

PVA

IPA imine

vioA

IPA imine dimer

Protoviolaceinic acid

Deoxychromoviridans

Violacein

vioB

vioE

vioD

vioC

vioC

I-tryptophan

PVA

IPA imine

vioA

IPA imine dimer

Protoviolaceinic acid

Deoxychromoviridans

Violacein

vioB

vioE

vioD

vioC

vioC

30oC

Ptet

RBS vioA vioB vioE RBS RBS

tetR

Plac

vioD 370 RBS

370 RBS

tetR

Plac

vioD 370 RBS

370 RBS

C.

A.

B.

vioC RBS lacI RBS tetR RBS

42oC induced Device

J23114

RBS CI

PcI

30oC

30oC

vioC RBS lacI RBS tetR

J23114

RBS CI

PcI

RBS

PVA

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I-tryptophan

PVA

IPA imine

vioA

IPA imine dimer

Protoviolaceinic acid

Deoxychromoviridans

Violacein

vioB

vioE

vioD

vioC

vioC

30oC42oC

Ptet

RBS vioA vioB vioE RBS RBS

tetR

Plac

vioD 370 RBS

370 RBS

42 oC

Deoxychromoviridans

30oC

42oC

Ptet

RBS vioA vioB vioE RBS RBS

tetR

Plac

vioD 370 RBS

370 RBS

C.

A.

B.

vioC RBS lacI RBS tetR RBS

42oC induced Device

J23114

RBS CI

PcI

lacI tet

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Protoviolaceinic acid

I-tryptophan

Protodeoxyviolaceinic acid(PVA)

IPA imine

vioA

IPA imine dimer

Deoxychromoviridans

Violacein

vioB

vioE

vioD

vioC

vioC

Violancein Pathway

37oC

42oC

30⁰C

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Drug Achivements

Achievements

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Designed “Pathway Commander”

Carotenoid synthesis pathway : BBa_K539151 and BBa_K539281

BBa_K539151 and BBa_K539281

Achievements

Designed “Pathway Commander”

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Achievements

Carotenoid synthesis pathway : BBa_K539151 and BBa_K539281

Designed “Pathway Commander”

Isobutanol pathway : BBa_K539691 and BBa_K539742

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Achievements

BBa_K118008

RBS vioA vioC vioB vioE RBS RBS RBS

BBa_K274004

Correct point mutation : BBa_K118008 and BBa_K274004

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Achievements

BBa_K098995

BBa_K115002

Characterize Biobrick (modeling) : BBa_K098995 and BBa_K115002

Correct point mutation : BBa_K118008 and BBa_K274004

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alsS

ilvC

ilvD

kivd

Achievements

Correct point mutation : BBa_K118008 and BBa_K274004

Characterize Biobrick (modeling) : BBa_K098995 and BBa_K115002

Submit novel Biobricks: alsS, ilvC, ilvD and kivd

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Submit 46 biobricks

Achievements

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Hsien-Da Huang Ching-Ping Tseng

Jenn-Kang Hwang Hsiao-Ching Lee

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Acknowledgement

We are NCTU Formosa !

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