water safe----providing you safe...
TRANSCRIPT
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Detection and degradation of Microcystin LR
By Jilin_China
Water safe----Providing you safe water
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Water
Safe
Jilin-China
Directory
Our team
Project
Human practice
Acknowledgement
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1Our
Team
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Team
We come from Jilin University,
Which is located in the north of China.
❶
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Mr.Wu
Mr.Gao
Ms.Jiang
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? ? ?
Advisor:Students:
Instructors:
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1 Our Team
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2 Project
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Project
Although we got dozens of ideas about the project and had heated discussions, we focused on one toxin in water pollution eventually.
❷
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Algae blooming in Lake Erie
• In this summer, Lake Erie has a worsening problem with toxic bacteria commonly known as blue-green algae.
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Contrast concentration of Microcystin LR:
Safe limit :1.045 µg/L—— The World Health Organization
Lake Erie :reaching 2.5 µg/L——Toledo-Lucas County Health Department
Terrible influence:
Microcystin LR was found in Toledo’s treated water, which caused 30,000 southern residents living without fresh water for two days.
Microcystin LR threatens water safety
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Here, comes the QUESTION:
Why we still work on it ?
More easy
More intelligentMore efficient
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So, what we do?
Find
1. We find the promoter which can detect the existence of MicrocystinLR in drinking water.
2. We construct a gene reporter which can express GFP to let us know.
3. We synthesized MlrA gene which can degrade Microcystin-LR in high efficient.
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A. How to find it ?
The Mlr gene cluster of Sphingonomonas sp.
Environ Toxicol. 2001;16(6):523-34
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Microcystin LR
?
We synthesized a RFP-promoter-GFP gene cluster to detect the relationship between MLR promoter and microcystin LR.
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The fragment between mlrC and mlrA contains a promoter, which can express GFP in E.coli and Lactococcus lactis Induced by Microcystin LR.
Result:
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B. How to report it?
GFP as a reporter
Mlrpromoter
pMG-mlr5482 bp
GFP
Mlr APromoter mlr
We put GFP gene into our system, which is situated upstream of Mlr A gene and downstream of Mlrpromoter.
When the Microcystin LR reaches the safe limit, we can know it by green fluorescence.
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C. How to degrade it?
Hydrolyse the tetrapeptide degradation product.Mlr C
Mlr A
Mlr D
Mlr B
Facilitate the transport of Microcystin LR or its degradation product.
Catalyse the degradation of linear Microcystin LR to a tetrapeptidedegradation product.
Cut the Microcystin LR cycle structure to a line.
Mlr C Mlr A Mlr D Mlr B
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Degraded by MlrA
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Degraded by MlrAToxin↓160 times!
Environ Toxicol. 2001;16(6):523-34
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Synthesized mlrA gene
The mlrA gene was synthesized according to the sequence of Sphingomonas sp. ACM-3962 containing 336 codons.
246 codons were optimized for increasing expression of mlrA in Lactococcus lactis.
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1. Fusion expression cluster of
mlr promoter-GFP-mlrA
2. Using E. coli-L. lactis shuttle expression vector pMG36e Expressed in E.coli and L.lactis.
3. L.lactis could avoid secondary pollution in the natural environment. Recombined vector
Achievements
pMG-mlr5482 bp
GFP
Mlr APromoter mlr
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M 1 2 M 3 4
The fusion protein GFP-mlrA was expressed in E.coliBL21 and it could be even more efficiently expressed in Lactococcus lactis.
E.coli L.lactis
97.4kD
66.2kD
43.0kD
31.0kD
M: Marker
1: non-induced in E.coli
2: induced in E.coli
3: non-induced in L.lactis
4: induced in L.lactis
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1E-3 0.005 0.01 0.05 0.1 0.5 1 5 10 --0
50
100
150
200R
ela
tiv
e f
luo
res
ce
nc
e i
nte
ns
ity
(%
)
Microcystin LR concentration (μg/L)
E.coli
Lactococcus lactis
Detection ability
The detection limit is 0.01 µg/L.
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Degradation ability
-2 0 2 4 6 8 10 12 14 16 18
0
2
4
6
8
10
Mic
roc
ysti
n L
R c
on
cen
tra
tio
n (
μg
/L)
Cultured time (h)
0.1 μg/L
1 μg/L
10 μg/L
-2 0 2 4 6 8 10 12 14 16 18
0
2
4
6
8
10
Mic
roc
ys
tin
LR
co
nc
en
tra
tio
n (
μg
/L)
Cultured time (h)
0.1 μg/L
1 μg/L
10 μg/L
E.coli L.lactis
Degradation: about 12 hrs at 10 µg/L;about 8 hrs at 1 µg/L
Degradation: about 16 hrs at 10 µg/L;about 8 hrs at 1 µg/L
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Practical application in future
Water Sample Freeze-dryingLactococcus lactis
Fluorescence Spectrophotometer
1-2 hrs
UV lamp bulb
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v
2 Project
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3 Human Practice
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Human Practice❸2014
Survey
Apr 15th
May 1st
Observation
Jun 6th
Cooperation
Aug 23rd
Communication
Propagate
Sep 15th
Nov 2nd
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A.Survey
We did a survey in the neighborhood community.
OBJECT: 2000 nearby lake’s residents
FORM: Questionnaire Survey
Conclusions:
1. Worried about drinking water and dozens of
complaints on the polluted water
2. Residents really want detection kits to test
drinking water in their home.
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A simple method to detect drinking water ?
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B. Field visits
In order to find the current situation about the algae bloom, we visited several lakes to find it out.
The South Lake in Changchun City
The SongHua Lake in Jilin City
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The South Lake in Changchun
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The SongHua Lake in Jilin
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C. Propagation
• To propagate and share iGEM, this great competition to more students, we prepare a special lecture to high school students from Affiliated Middle School of Jilin University.
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D. Communication
• NanJing University
----For communication and support
• Huazhong Agricultural University, Wuhan University and some other IGEM teams
---- For exchanging ideas
• Lanzhou University
----Assistance in theoretical analysis
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3. Human Practice
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Acknowledgement4
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Acknowledgement
It is our great honor to have your assistance.
❹
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We’d like to express our deepest thanks for individuals and
institutes generously giving help to us.
Laboratory supports:
Greatest thanks to National Engineering Laboratory AIDS
Vaccine (NELAV),
Translational Cancer Medicine Laboratory, Bio
pharmaceutical laboratory.Our experiments is totally
supported by them.
Material support:
Great thanks to Prof. Wu Yongge, Assoc Prof. Gao
Chaohui and Assoc Prof. Jiang Yuqun and all the
seniors and postgraduates in their labs.Without their
selfless help and endless materials, we couldn’t
accomplish the whole program.
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Welcome other team members!
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Q&A
Congratulation for IGEM’s 10th anniversary !