In vivo Evolution Engineering towards Tryptophan-overproduction Superbug iGEM2013 – Asia Regional Jamboree – Hong Kong – October 5, 2013
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
33
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
45
46
47
48