In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase

In vivo Evolution Engineering towards

Tryptophan-overproduction Superbug

iGEM2013 – Asia Regional Jamboree – Hong Kong – October 5, 2013

2

3

TryptophanProductivity

GenerationsAncestor

4

5

6

3’ to 5’ exonuclease activity lost

DNA Polymerase III

7

sfGFPRBS mutD

araBAD

RBS

0.0E+00

5.0E-09

1.0E-08

1.5E-08

2.0E-08

1 2 3 4

Mu

tati

on

ra

te(m

uta

tio

ns/

gen

era

tio

n)

pBAD_B0030-mutD-sfGFParabinose=1g/L

pBAD_ori_RBS-mutD-sfGFParabinose=1g/L

pBAD_B0030-mutD-sfGFParabinose=0g/L

pBAD-sfGFParabinose=0g/L

1 2

3 4

Part Name: BBa_K1025003

8

9

P site A site

𝑹𝒉𝒐 𝒇𝒂𝒄𝒕𝒐𝒓

𝑇𝑟𝑦𝑝𝑡𝑜𝑝ℎ𝑎𝑛

GGA

𝑇𝑛𝑎𝐶 − 𝑝𝑒𝑝𝑡𝑖𝑑𝑦𝑙 − 𝑡𝑅𝑁𝐴𝑃𝑟𝑜

𝑝𝑒𝑝𝑡𝑖𝑑𝑦𝑙𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑎𝑠𝑒 𝑐𝑒𝑛𝑡𝑒𝑟

······ ··· ··· ··· ··· ··· ··· ··· ··· CGC CCU UGA ··· rut site ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· mRNA𝒕𝒓𝒂𝒏𝒔𝒄𝒓𝒊𝒑𝒕𝒊𝒐𝒏𝒕𝒆𝒓𝒎𝒊𝒏𝒂𝒕𝒊𝒐𝒏 𝒔𝒊𝒕𝒆𝒔

𝑏𝑜𝑥𝐴 𝑠𝑒𝑞𝑢𝑒𝑛𝑐𝑒𝑡𝑛𝑎𝐶 𝑠𝑡𝑜𝑝 𝑐𝑜𝑑𝑜𝑛

Gong, F. & Yanofsky, C. Instruction of translating ribosome by nascent peptide.Science 297, 1864-1867, doi:10.1126/science.1073997 (2002) 10

StoplacZRBS tnaC-Rho-tnaA

Ptac

RBS

Trp

Working Solution Working SolutionWorking Solution

Part Name: BBa_K1025006

11

Dynamic range0 – 3mM

Activity of beta-lactamase against tryptophan level

0-3.0 mMlinear relationship

12

13

StoptetAtnaC-Rho-tnaA

Ptac

tetAtnaC-Rho-tnaA

14

Part Name: BBa_K1025007

RBSRBS

Trp

15

16

ResultBitter Defender

Trp 000(Trp production 0mg/L)

tetracycline= 80 mg/L

tetracycline= 0 mg/L

tetracycline= 160 mg/L

tetracycline= 120 mg/L

Trp 007(Trp production 70mg/L)

Trp 015(Trp production 150mg/L)

RBS Screening:RBS_ori: AGGAAACAGACCATGGCT

RBS_B0030: GAAAATGAAACCCAACARBS_B0032: AGATGAAACCCAACATA

0

0.5

1

1.5

2

0 10 20 30

OD

60

0

t/h

0

0.5

1

1.5

2

0 10 20 30O

D6

00

t/h

0

0.5

1

1.5

2

0 10 20 30

OD

60

0

t/h

0

0.5

1

1.5

2

0 10 20 30

OD

60

0

t/h

tetracycline = 0 mg/Ltetracycline = 80 mg/L

tetracycline = 120 mg/L tetracycline = 160 mg/L

17

18

StopmalQRBS tnaC-Rho-tnaA

araBAD

RBS

malQ

malQGenome malQ Knock-out

malQtnaC-Rho-tnaA

19

Part Name: BBa_K1025008

Trp

20

21

RBS_3-4 JW 3379(∆ 𝑚𝑎𝑙Q)

mal= 4 g/L

mal= 2 g/L

mal= 8 g/L

mal= 6 g/L

Result

RBS Screening: RRRRRRNNNNNNN

RBS_3-4: AAAAAAATGTGCT

22

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0 2 4 6 8 10 12 14 16

OD

time/h

strain 3-4 growth curve in 4g/L maltose M9 media

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

0 2 4 6 8 10 12 14 16

OD

time/h

strain 3-4 growth curve in 6g/L maltose M9 media

Mutator - mutD

- 10 fold increase in mutation rate

Detector – Tryptophan sensor

- Tryptophan dynamic range 0 – 3mM

Selector 1 – bitter defender part

- Tryptophan dependent growth

Selector 2 – sweet pressure part

- Tryptophan dependent growth

23

1st generation 2nd generation 3rd generation

tetracycline = 120mg/L

dilution dilution

Ancestor StrainControl Strain

1

Control Strain

2

(E.coli BL21)

24

Evolving Strain

tetracycline = 160mg/L tetracycline = 200mg/L

EvolvingStrain

ControlStrain1

ControlStrain2

Growth conditions(OD600)1st

generation

2nd

generation

3rd

generation

0.917 0.342 0.442

0.69 0.697 0.581

No growth No growth No growth

25

0

1

2

3

4

5

6

7

8

9

10

0 (ancestor) 1 2 3

Try

pto

ph

an

(m

g/L

/OD

)

Generations

Titer increase dynamics after different rounds of evolution

bitter defender part/mut part

bitter defender part

26

0

1

2

3

4

5

6

7

8

9

10

0 (ancestor) 1 2 3

Try

pto

ph

an

(m

g/L

/OD

)

Generations

Titer increase dynamics after different rounds of evolution

Plating

Fermentation

27

0.03

0.42

1.14

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Ancestor Control strain 1 Evolving strain

Trp

top

ho

n (

mg

/L)

28

TryptophanProductivity

GenerationsAncestor

29

30

31

BIO-FARM

BACTERIA PAINTING

LECTURE

32FILM

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34

35

Auxin System

Imperial College

London iGEM 2011

help seeds fight

degradation

Bio-decontamination Kit

PKU 2010

detect and absorb heavy

metals in aquatic

AgrEcoli

BCCS-Bristol in

2010

soil fertility sensor

E.glowli

Cambridge

IGEM 2010

bright light

output in a

range of

different

colours

Flower

Fairy E.coli

Kyoto

IGEM 2012

help plants

bloom

SynBiological Bug

Buster

HKUST in 2009

detect, attract and

eliminate pests

Edinburgh

cooker

Edinburgh

IGEM 2008

produce

starch from

the

cellulose 36

37

38

39

40

41

42

43

BIO-FARM

BACTERIA PAINTING

LECTURE

44FILM

• Professor Xinhui Xing

• Prefessor Chong Zhang

• PhD candidate Xiang ZHENG

• PhD candidate Mingyue FANG

• Jili Bai Zhenlong Wei

• SU Hang Tianyi You

• Biomed

• E.coli Kello Knockout Collection

• Taihe Biotechnology Co.,LTD

• Life Technologies Corporation

• Bio-Rad Laboratories

• Fundamental Industry Training

Center of Tsinghua University

• Green Industry biotechnology

Lab

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