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

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

❶ Team We come from Jilin University, Which is located in the north of China.

Ms. Mr. Mr. Jiang Wu Gao

Advisor : Students: Instructors: ? ? ?

Our Team 1

2 Project

❷ Project Although we got dozens of ideas about the project and had heated discussions, we focused on one toxin in water pollution eventually.

Algae blooming in Lake Erie • In this summer, Lake Erie has a worsening problem with toxic bacteria commonly known as blue-green algae.

Microcystin LR threatens water safety  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.

Here, comes the QUESTION: Why we still work on it ? More easy More intelligent More efficient

So, what we do? 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 Find know. 3. We synthesized MlrA gene which can degrade Microcystin-LR in high efficient.

A. How to find it ? The Mlr gene cluster of Sphingonomonas sp. Environ Toxicol . 2001;16(6):523-34

Microcystin LR ? We synthesized a RFP-promoter-GFP gene cluster to detect the relationship between MLR promoter and microcystin LR.

Result: The fragment between mlrC and mlrA contains a promoter, which can express GFP in E.coli and Lactococcus lactis Induced by Microcystin LR.

B. How to report it? We put GFP gene into our GFP as a reporter system, which is situated upstream of Mlr A gene and Mlr downstream of Mlr promoter promoter. GFP When the Microcystin LR Mlr A Promoter mlr reaches the safe limit, we pMG-mlr can know it by green 5482 bp fluorescence.

C. How to degrade it? Mlr C Mlr A Mlr D Mlr B Mlr C Hydrolyse the tetrapeptide degradation product. Cut the Microcystin LR cycle structure to a line. Mlr A Facilitate the transport of Microcystin LR or its degradation product. Mlr D Catalyse the degradation of linear Microcystin LR to a tetrapeptide Mlr B degradation product.

Degraded by MlrA

Toxin ↓ Degraded by MlrA 160 times! Environ Toxicol . 2001;16(6):523-34

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 .

Achievements 1. Fusion expression cluster of mlr promoter-GFP-mlrA GFP Mlr A Promoter mlr 2. Using E. coli-L. lactis shuttle pMG-mlr expression vector pMG36e 5482 bp Expressed in E.coli and L.lactis . 3. L.lactis could avoid secondary pollution in the natural Recombined vector environment.

M 1 2 M 3 4 M: Marker 97.4kD 1: non-induced in E.coli 66.2kD 2: induced in E.coli 43.0kD 3: non-induced in L.lactis 4: induced in L.lactis 31.0kD E.coli L.lactis The fusion protein GFP-mlrA was expressed in E.coli BL21 and it could be even more efficiently expressed in Lactococcus lactis.

Detection ability Relative fluorescence intensity (%) 200 E.coli Lactococcus lactis 150 100 50 0 1E-3 0.005 0.01 0.05 0.1 0.5 1 5 10 -- Microcystin LR concentration (μg/L) The detection limit is 0.01 µg/L.

Degradation ability Microcystin LR concentration (μg/L) 0.1 μg/L 0.1 μg/L Microcystin LR concentration (μg/L) 10 10 1 μg/L 1 μg/L 10 μg/L 10 μg/L 8 8 6 6 4 4 2 2 0 0 -2 0 2 4 6 8 10 12 14 16 18 -2 0 2 4 6 8 10 12 14 16 18 Cultured time (h) Cultured time (h) E.coli L.lactis Degradation: about 16 hrs at 10 µg/L; Degradation: about 12 hrs at 10 µg/L; about 8 hrs at 1 µg/L about 8 hrs at 1 µg/L

Practical application in future Freeze-drying Water Sample Lactococcus lactis UV lamp bulb 1-2 hrs Fluorescence Spectrophotometer

2 Project v

3 Human Practice

❸ Human Practice 2014 Observation Communication Jun 6 th Sep 15 th Apr 15 th May 1 st Aug 23 rd Nov 2 nd Survey Cooperation Propagate

A.Survey We did a survey in the neighborhood community. OBJECT: 2000 n earby 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.

A simple method to detect drinking water ？

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

The South Lake in Changchun

The SongHua Lake in Jilin

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.

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

3. Human Practice

4 Acknowledgement

❹ Acknowledgement It is our great honor to have your assistance.

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 onal al Engineeri ering g Laborat atory ory AIDS DS Vaccine ne (NE NELAV AV), ), Translat nslation onal 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 orted by them. em. Mater terial l support: ort: Great eat thanks ks to Prof. f. Wu Yongge, , Assoc c Prof. f. Gao Ch Chaohui and As Assoc oc 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 out their r selfless help and endless materials, we couldn’t accompl mplish sh the wh whole program ram.

Welcome other team members!

Q&A Congratulation for IGEM’s 10 th anniversary !