Tryptophan-overproduction Superbug iGEM2013 Asia Regional Jamboree - - PowerPoint PPT Presentation

In vivo Evolution Engineering towards Tryptophan-overproduction Superbug

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

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Tryptophan Productivity Generations

Ancestor

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3’ to 5’ exonuclease activity lost DNA Polymerase III 7

sfGFP

RBS

mutD

araBAD RBS 0.0E+00 5.0E-09 1.0E-08 1.5E-08 2.0E-08

1 2 3 4 Mutation rate(mutations/generation) pBAD_B0030-mutD-sfGFP arabinose=1g/L pBAD_ori_RBS-mutD-sfGFP arabinose=1g/L pBAD_B0030-mutD-sfGFP arabinose=0g/L pBAD-sfGFP arabinose=0g/L

1 2 3 4

Part Name: BBa_K1025003

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P sit site A A sit site 𝑺𝒊𝒑 𝒈𝒃𝒅𝒖𝒑𝒔

𝑈𝑠𝑧𝑞𝑢𝑝𝑞ℎ𝑏𝑜 GG GGA 𝑈𝑜𝑏𝐷 − 𝑞𝑓𝑞𝑢𝑗𝑒𝑧𝑚 − 𝑢𝑆𝑂𝐵𝑄𝑠𝑝 𝑞𝑓𝑞𝑢𝑗𝑒𝑧𝑚𝑢𝑠𝑏𝑜𝑡𝑔𝑓𝑠𝑏𝑡𝑓 𝑑𝑓𝑜𝑢𝑓𝑠 ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· CGC CCU UGA ··· rut site ··· ··· ··· ··· ··· ··· ··· ··· ··· ···

𝒖𝒔𝒃𝒐𝒕𝒅𝒔𝒋𝒒𝒖𝒋𝒑𝒐 𝒖𝒇𝒔𝒏𝒋𝒐𝒃𝒖𝒋𝒑𝒐 𝒕𝒋𝒖𝒇𝒕

𝑐𝑝𝑦𝐵 𝑡𝑓𝑟𝑣𝑓𝑜𝑑𝑓 𝑢𝑜𝑏𝐷 𝑡𝑢𝑝𝑞 𝑑𝑝𝑒𝑝𝑜

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

Stop lacZ RBS tnaC-Rho-tnaA

Ptac

RBS

Trp Working Solution Working Solution Working Solution

Part Name: BBa_K1025006

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Dynamic range 0 – 3mM

Activity of beta-lactamase against tryptophan level

0-3.0 mM linear relationship

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Stop tetA tnaC-Rho-tnaA

Ptac

tetA

tnaC-Rho-tnaA

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Part Name: BBa_K1025007

RBS RBS

Trp

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Result

Bitter 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: GAAAATGAAACCCAACA RBS_B0032: AGATGAAACCCAACATA

0.5 1 1.5 2 10 20 30

OD600 t/h

0.5 1 1.5 2 10 20 30

OD600 t/h

0.5 1 1.5 2 10 20 30

OD600 t/h

0.5 1 1.5 2 10 20 30

OD600 t/h

tetracycline = 0 mg/L tetracycline = 80 mg/L

tetracycline = 120 mg/L

tetracycline = 160 mg/L

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Stop malQ RBS tnaC-Rho-tnaA

araBAD

RBS

malQ malQ Genome malQ Knock-out malQ tnaC-Rho-tnaA 19

Part Name: BBa_K1025008

Trp

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RBS_3-4 JW 3379

(∆ 𝑛𝑏𝑚Q)

mal= 4 g/L mal= 2 g/L mal= 8 g/L mal= 6 g/L

Result

RBS Screening: RRRRRRNNNNNNN

RBS_3-4: AAAAAAATGTGCT

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0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2 4 6 8 10 12 14 16 OD time/h

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

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 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

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1st generation 2nd generation 3rd generation tetracycline = 120mg/L dilution dilution

Ancestor Strain Control Strain

1

Control Strain

2 (E.coli BL21)

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Evolving Strain

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

Evolving

Strain

Control

Strain1

Control

Strain2

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

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1 2 3 4 5 6 7 8 9 10 0 (ancestor) 1 2 3 Tryptophan (mg/L/OD) Generations

Titer increase dynamics after different rounds of evolution

bitter defender part/mut part bitter defender part

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1 2 3 4 5 6 7 8 9 10 0 (ancestor) 1 2 3

Tryptophan (mg/L/OD)

Generations Titer increase dynamics after different rounds of evolution

Plating Fermentation 27

0.03 0.42 1.14

0.2 0.4 0.6 0.8 1 1.2 1.4

Ancestor Control strain 1 Evolving strain Trptophon (mg/L)

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Tryptophan Productivity Generations

Ancestor 29

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BIO-FARM BACTERIA PAINTING LECTURE 32 FILM

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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

• utput 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

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BIO-FARM BACTERIA PAINTING LECTURE 44 FILM

• 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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