In vivo Evolution Engineering towards Tryptophan...
Transcript of In vivo Evolution Engineering towards Tryptophan...
![Page 1: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/1.jpg)
In vivo Evolution Engineering towards
Tryptophan-overproduction Superbug
iGEM2013 – Asia Regional Jamboree – Hong Kong – October 5, 2013
![Page 2: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/2.jpg)
2
![Page 3: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/3.jpg)
3
![Page 4: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/4.jpg)
TryptophanProductivity
GenerationsAncestor
4
![Page 5: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/5.jpg)
5
![Page 6: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/6.jpg)
6
![Page 7: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/7.jpg)
3’ to 5’ exonuclease activity lost
DNA Polymerase III
7
![Page 8: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/8.jpg)
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
![Page 9: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/9.jpg)
9
![Page 10: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/10.jpg)
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
![Page 11: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/11.jpg)
StoplacZRBS tnaC-Rho-tnaA
Ptac
RBS
Trp
Working Solution Working SolutionWorking Solution
Part Name: BBa_K1025006
11
![Page 12: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/12.jpg)
Dynamic range0 – 3mM
Activity of beta-lactamase against tryptophan level
0-3.0 mMlinear relationship
12
![Page 13: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/13.jpg)
13
![Page 14: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/14.jpg)
StoptetAtnaC-Rho-tnaA
Ptac
tetAtnaC-Rho-tnaA
14
Part Name: BBa_K1025007
RBSRBS
Trp
![Page 15: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/15.jpg)
15
![Page 16: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/16.jpg)
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
![Page 17: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/17.jpg)
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
![Page 18: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/18.jpg)
18
![Page 19: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/19.jpg)
StopmalQRBS tnaC-Rho-tnaA
araBAD
RBS
malQ
malQGenome malQ Knock-out
malQtnaC-Rho-tnaA
19
Part Name: BBa_K1025008
Trp
![Page 20: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/20.jpg)
20
![Page 21: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/21.jpg)
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
![Page 22: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/22.jpg)
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
![Page 23: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/23.jpg)
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
![Page 24: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/24.jpg)
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
![Page 25: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/25.jpg)
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
![Page 26: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/26.jpg)
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
![Page 27: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/27.jpg)
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
![Page 28: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/28.jpg)
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
![Page 29: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/29.jpg)
TryptophanProductivity
GenerationsAncestor
29
![Page 30: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/30.jpg)
30
![Page 31: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/31.jpg)
31
![Page 32: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/32.jpg)
BIO-FARM
BACTERIA PAINTING
LECTURE
32FILM
![Page 33: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/33.jpg)
33
![Page 34: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/34.jpg)
34
![Page 35: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/35.jpg)
35
![Page 36: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/36.jpg)
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
![Page 37: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/37.jpg)
37
![Page 38: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/38.jpg)
38
![Page 39: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/39.jpg)
39
![Page 40: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/40.jpg)
40
![Page 41: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/41.jpg)
41
![Page 42: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/42.jpg)
42
![Page 43: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/43.jpg)
43
![Page 44: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/44.jpg)
BIO-FARM
BACTERIA PAINTING
LECTURE
44FILM
![Page 45: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/45.jpg)
• 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
![Page 46: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/46.jpg)
46
![Page 47: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/47.jpg)
47
![Page 48: In vivo Evolution Engineering towards Tryptophan ...2013.igem.org/files/presentation/Tsinghua-E.pdf · Detector –Tryptophan sensor ... (ancestor) 1 2 3 Generations Titer increase](https://reader034.fdocuments.us/reader034/viewer/2022052008/601d2637a8327676a457072f/html5/thumbnails/48.jpg)
48