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 4
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DNA Polymerase III 3’ to 5’ exonuclease activity lost 7
ara BAD 1 2 RBS RBS sfGFP mut D Part Name: BBa_K1025003 2.0E-08 rate(mutations/generation) pBAD_ori_RBS-mutD-sfGFP pBAD_B0030-mutD-sfGFP 1.5E-08 arabinose=1g/L arabinose=1g/L Mutation 3 4 1.0E-08 5.0E-09 0.0E+00 1 2 3 4 pBAD- sfGFP pBAD_B0030-mutD-sfGFP 8 arabinose=0g/L arabinose=0g/L
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𝑈𝑜𝑏𝐷 − 𝑞𝑓𝑞𝑢𝑗𝑒𝑧𝑚 − 𝑢𝑆𝑂𝐵 𝑄𝑠𝑝 𝑈𝑠𝑧𝑞𝑢𝑝𝑞ℎ𝑏𝑜 𝑞𝑓𝑞𝑢𝑗𝑒𝑧𝑚𝑢𝑠𝑏𝑜𝑡𝑔𝑓𝑠𝑏𝑡𝑓 𝑑𝑓𝑜𝑢𝑓𝑠 𝑺𝒊𝒑 𝒈𝒃𝒅𝒖𝒑𝒔 GG GGA 𝒖𝒔𝒃𝒐𝒕𝒅𝒔𝒋𝒒𝒖𝒋𝒑𝒐 ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· CGC CCU UGA ··· rut site ··· ··· ··· ··· ··· ··· ··· ··· ··· ··· 𝒖𝒇𝒔𝒏𝒋𝒐𝒃𝒖𝒋𝒑𝒐 𝒕𝒋𝒖𝒇𝒕 P sit site A A sit site 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
Ptac tna C- Rho - tna A Stop RBS RBS lac Z Part Name: BBa_K1025006 Working Solution Working Solution Working Solution Trp 11
0-3.0 mM Dynamic range linear relationship 0 – 3mM Activity of beta-lactamase against tryptophan level 12
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Ptac tet A Stop tna C-Rho- tna A RBS RBS Part Name: BBa_K1025007 tna C-Rho -tna A tet A Trp 14
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RBS Screening: RBS_ori: AGGAAACAGACCATGGCT RBS_B0030: GAAAATGAAACCCAACA RBS_B0032: AGATGAAACCCAACATA Trp 000 tetracycline= 0 mg/L (Trp production 0mg/L) tetracycline= 80 mg/L Result Trp 007 Bitter (Trp production 70mg/L) tetracycline= 120 mg/L Defender Trp 015 tetracycline= 160 mg/L (Trp production 150mg/L) 16
tetracycline = 0 mg/L 2 2 tetracycline = 80 mg/L 1.5 1.5 OD 600 OD 600 1 1 0.5 0.5 0 0 0 10 20 30 0 10 20 30 t/h t/h 2 2 tetracycline = 160 mg/L tetracycline = 120 mg/L 1.5 1.5 OD 600 OD 600 1 1 0.5 0.5 0 0 0 10 20 30 0 10 20 30 17 t/h t/h
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ara BAD RBS tna C-Rho- tna A RBS mal Q Stop Part Name: BBa_K1025008 Trp tna C-Rho- tna A mal Q malQ malQ Genome ma lQ Knock-out 19
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RBS Screening: RRRRRRNNNNNNN RBS_3-4: AAAAAAATGTGCT mal= 2 g/L mal= 4 g/L JW 3379 Result RBS_3-4 ( ∆ 𝑛𝑏𝑚Q ) mal= 6 g/L mal= 8 g/L 21
strain 3-4 growth curve in 4g/L strain 3-4 growth curve in 6g/L maltose M9 media maltose M9 media 2.2 2.2 2 2 1.8 1.8 1.6 1.6 1.4 1.4 1.2 1.2 OD OD 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 time/h time/h 22
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
Control Strain ( E.coli BL21) Ancestor Strain 1 Contro l Strain Evolving Strain 2 1st generation 2nd generation 3rd generation dilution dilution tetracycline = 120mg/L tetracycline = 160mg/L tetracycline = 200mg/L 24
1st 2nd 3rd Growth conditions(OD600) generation generation generation Evolving 0.917 0.342 0.442 Strain Control 0.69 0.697 0.581 Strain1 Control No growth No growth No growth Strain2 25
Titer increase dynamics after different rounds of evolution 10 9 8 Tryptophan (mg/L/OD) 7 bitter defender part/mut part 6 5 4 bitter defender part 3 2 1 0 0 (ancestor) 1 2 3 Generations 26
Plating Titer increase dynamics after different rounds of evolution 10 9 8 Tryptophan (mg/L/OD) 7 Fermentation 6 5 4 3 2 1 0 0 (ancestor) 1 2 3 Generations 27
1.4 1.14 1.2 1 Trptophon (mg/L) 0.8 0.6 0.42 0.4 0.2 0.03 0 Ancestor Control strain 1 Evolving strain 28
Tryptophan Productivity Generations Ancestor 29
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BIO-FARM LECTURE BACTERIA PAINTING FILM 32
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Auxin System Bio-decontamination Kit Imperial College SynBiological Bug AgrEcoli PKU 2010 London iGEM 2011 Buster BCCS-Bristol in detect and absorb heavy help seeds fight HKUST in 2009 2010 metals in aquatic degradation detect, attract and soil fertility sensor eliminate pests E.glowli Flower Cambridge Edinburgh Fairy E.coli IGEM 2010 cooker Kyoto bright light Edinburgh IGEM 2012 output in a IGEM 2008 help plants range of produce bloom different starch from colours the cellulose 36
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BIO-FARM LECTURE BACTERIA PAINTING FILM 44
• • Professor Xinhui Xing Taihe Biotechnology Co.,LTD • • Life Technologies Corporation Prefessor Chong Zhang • • Bio-Rad Laboratories PhD candidate Xiang ZHENG • • Fundamental Industry Training PhD candidate Mingyue FANG Center of Tsinghua University • Jili Bai Zhenlong Wei • Green Industry biotechnology • SU Hang Tianyi You Lab • Biomed • E.coli Kello Knockout Collection 45
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