Water safe ----Providing you safe water Detection and degradation - - PowerPoint PPT Presentation

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Water safe ----Providing you safe water Detection and degradation - - PowerPoint PPT Presentation

Mar 27, 2023 235 likes •663 views

Water safe ----Providing you safe water Detection and degradation of Microcystin LR By Jilin_China Directory Our team Water Project Safe Human practice Jilin-China Acknowledgement Our 1 Team Team We come from Jilin University,

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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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1

Our

Team

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Team

We come from Jilin University, Which is located in the north

  • f 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

  • n 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 intelligent More efficient

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So, what we do? Find

  • 1. We find the promoter which can

detect the existence of Microcystin LR 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 Mlr promoter

pMG-mlr

5482 bp

GFP Mlr A Promoter mlr

We put GFP gene into our system, which is situated upstream of Mlr A gene and downstream of Mlr promoter. 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 tetrapeptide degradation 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 MlrA

Toxin↓ 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-mlr

5482 bp

GFP Mlr A Promoter mlr
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M 1 2 M 3 4 The fusion protein GFP-mlrA was expressed in E.coli BL21 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

  • 50

100 150 200

Relative fluorescence intensity (%) 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
2 4 6 8 10 12 14 16 18 2 4 6 8 10

Microcystin LR concentration (μg/L) Cultured time (h)

0.1 μg/L 1 μg/L 10 μg/L
  • 2
2 4 6 8 10 12 14 16 18 2 4 6 8 10

Microcystin LR concentration (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-drying Lactococcus 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 Practice

❸

2014

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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Acknowledgement

4

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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 institut titutes s genero rously usly giving help to us. Laborator ratory y supports rts： Greatest eatest thanks nks to Na Nation

  • nal

al Engineeri ering g Laborat atory

  • ry AIDS

DS Vaccine ne (NE NELAV AV), ), Translat nslation

  • nal

al Ca Cancer r Medicine ine Laboratory, atory, Bio pharmace maceut utica cal l laborator ratory. y.Our Our experime riments nts is total tally y supported

  • rted by them.

em. Mater terial l support:

  • rt:

Great eat thanks ks to Prof.

  • f. Wu Yongge,

, Assoc c Prof.

  • f. Gao

Ch Chaohui and As Assoc

  • c Pr

Prof.

  • f. Jia

iang Yu Yuqun and all ll the seniors rs and postgr tgrad aduates uates in their r labs.Wi .With thout

  • ut their

r selfless help and endless materials, we couldn’t accompl mplish sh the wh whole program ram.

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Welcome other team members!

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Q&A

Congratulation for IGEM’s 10th anniversary !