2014 iGEM 1 NEFU-China Northeast Forestry University 2 N orth E - - PowerPoint PPT Presentation

2014

iGEM

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NEFU-China

Northeast Forestry University

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NEFU Nanocrystal E.coli Flocculation Units N E F U NorthEast Forestry University

Inspiration

Shocking News：

http://news.163.com/13/0731/08/953M00U80001124J_all.html

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Shocking News：

The water in northeastern Indian state

• f Assam showed a high level of
• cadmium. Bad drinking water in this

place can cause immense health issues for people, and this problem is even more apparent with children. And thus may threaten the region’s youth and future generations.

• Two million people at risk of death from poisoned water

Cadmium Pollution – A Serious Concern

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Inspiration

Soil cadmium contents in 30 provinces of China

Cd/mg．kg-1

China environmental quality standard for soil GB15618-1995

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Inspiration

0~0.025 0.025~0.077 0.077~0.21 0.21~0.39 0.39~0.59 0.59~0.91 0.91~1.32 1.32~1.88

Cadmium Pollution – A Serious Concern

Visiting Sewage Plant

China environmental quality standard for water GB8978-1996

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Inspiration

Inductively Coupled Plasma - Optical Emission Spectrometry

Automobile Exhaustion Industrial Waste Pesticides & Fertilizers Non-ferrous metal smelting

Sources

Ni-Cd Batteries

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Diseases

Cancer Itai-Itai Disease Anemia Pneumonia Diabetes

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• Which is the best way to deal with heavy metal pollution?
• What kind of problems will be triggered by biological treatment?

SURVEY

185 respondents (93 male, 92 female)

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Ethical issues

Module 1 Detecting Module 2 Recycling Module 3 Flocculating

Detect the presence of Cadmium ions Synthesize the CdS nanocrystals Collect the nanocrystals by flocculating 10 10

Our Design

Which is suitable in our Project?

System Members Metal sensed Host

Mer R Mer R Hg

Prokaryotes

Pbr R Pb

Prokaryotes

Ars R/Smt B Ars R As,Sb

Prokaryotes

Smt B Zn,Cd

Prokaryotes

Nik R Nik R Ni

Prokaryotes

……

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Pennella, M. A. Giedroc Biometals, 2005.

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

Stop Stop smtO-P smtB smtA

S.D. S.D.

• 10(smtB)
• 10(smtA)

Inverted repeat Inverted repeat

smtB smtA

Huckle, J. W. Mol Microbiol, 1993.

12 12 Synechococcus PCC7942 smt-locus structure

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

13 13 Stop Stop smtO-P smtB smtA

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

Huckle, J. W. Mol Microbiol, 1993.

Cd2+

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pACYC184

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

MCS

Huckle, J. W. Mol Microbiol, 1993.

smtA Stop Stop smtO-P smtB

Cd2+

amilCP

Time (h) OD540

smtB-OP-smtA (+) Control

1.2 1.0 0.8 0.6 0.4 0.2 0.0 20 40 60 80 100 120 140 1.4

Results

smt-locus (+) strain: more tolerant of Cd2+

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Growth of cells containing smt-locus element in LB medium supplemented with 2μM Cd2+

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

More sensitive to Cd2+ with shorter period than Zn2+

Results

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Metal-induced expression of the pigments Cd2+ and Zn2+ (2μM) supplement for 1-12h Cd2+ and Zn2+ (1-50μM) supplement for 2h

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM Cd2+ Zn2+

Concentration (μM)

2 5 10 20 50 2.0 1.5 1.0 0.5 0.0

OD450 Time (h) OD450

2.5 2.0 1.5 1.0 0.5 0.0 2 4 6 8 10 12 Cd2+ Zn2+

3.0

MODELING FLOCCULATING SYSTEM RECYCLING SYSTEM DETECTING SYSTEM

Visualized Output

Results

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500 200 100 50 20 10 Saturation

(μM) (μM) (μM) (μM) (μM) (μM)

smtO-P smtB amilCP

Cd2+ ① ② ③

Stop Stop

Cd2+ Cd2+

Equations

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MODELING FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM

dp = r1(P - p) dX/dt = x0(1-f(p)) dY/dt = y0(1-X/x0t)

Equations

Simplifying the equation (4), we work out the equation (5). (4) Y= y0tf(P – etr1) (5) Y= y0tf(P)

Y y0 p P r1 Expression level of reporter gene The maximum amount per unit time Concentration of Cd2+ in extracellular Concentration of Cd2+ in cell The moving rate that Cd2+ enter into cells

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MODELING FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM

Fig.2 Cdmium-induced expression of the pigment (amilCP) with different concentrations Fig.1 Cdmium-induced expression of the pigment (amilCP) with a constant concentration(1μM)

Data

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MODELING FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM 2.0 1.5 1.0 0.5 0.0 Time (h) OD600 2.0 1.5 1.0 0.5 0.0

Reporter expression

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MODELING FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM

Fit 1 Y = 1.021 - 0.0003145P2 + 0.04481P RMSE=0.3657 R2=0.8739 Reporter expression Fit 1

Fig.3

Concentration (μM)

20 120 60 80 100 40

OD600

2.0 1.5 1.0 0.5 0.0 2.5 3.0

Fit 2 Fit 2 Y = 3.063 – 2.321P-0.2802 RMSE=0.0589 R2=0.9967

Module 1 Detecting Module 2 Recycling Module 3 Flocculating

Detect the presence of Cadmium ions Synthesize the CdS nanocrystals Collect the nanocrystals by flocculating 22 22

Our Design

MCS

RECYCLING SYSTEM FLOCCULATING SYSTEM MODELING DETECTING SYSTEM

Jensen, P.R. Appl Environ Microbiol, 1998.

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pET28b

CdS Nano S2- Short Peptide Cd2+ Stop CDS7 CP25

0.2μm

CdS Nano

+

CdCl2 + Na2S CdS CdS Short Peptide

MCS

RECYCLING SYSTEM FLOCCULATING SYSTEM MODELING DETECTING SYSTEM

Jensen, P.R. Appl Environ Microbiol, 1998.

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pET28b

CdS Nano S2- Short Peptide Cd2+ Stop CDS7 CP25 CdS Nano

+

CdCl2 + Na2S CdS CdS Short Peptide

MCS

RECYCLING SYSTEM FLOCCULATING SYSTEM MODELING DETECTING SYSTEM

Jensen, P.R. Appl Environ Microbiol, 1998.

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pET28b

CdS Nano S2- Short Peptide Cd2+ Stop CDS7 CP25

0.2μm

One E.coli cell (TEM)

Module 1 Detecting Module 2 Recycling Module 3 Flocculating

Our Design

Detect the presence of Cadmium ions Synthesize the CdS nanocrystals Collect the nanocrystals by flocculating 26 26

MCS

Fang Ma, China water and wastewater, 2003.

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pET28b

FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM MODELING

IPTG

T7 promoter Stop Flocculation gene

MCS

Fang Ma, China water and wastewater, 2003.

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pET28b

FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM MODELING

IPTG

T7 promoter Stop Flocculation gene

FLOCCULATING SYSTEM DETECTING SYSTEM RECYCLING SYSTEM MODELING

Improved sediment ability

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Results

Floc.（+） Floc.（-） 100 80 60 40 20 5 10 15 20

Settling Time (min) Settling Cells (%)

Module 1 Detecting Module 2 Recycling Module 3 Flocculating

Summary

Detect the presence of Cadmium ions Synthesize the CdS nanocrystals Collect the nanocrystals by flocculating 30 30

0.2μm

Achievement

We submitted four functional parts:

BBa_K1509000 BBa_K1509001 BBa_K1509002 BBa_K1509003

Conferences Online survey Visiting sewage plant

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• Parts
• Collaboration
• Human practice

Team ZJU-China Team HIT-Harbin

Human Practice

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Acknowledgement

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Northeast Forestry University Harbin Institute of Technology National Natural Science Foundation of China School of Forestry, Northeast Forestry University School of Science, Northeast Forestry University College of Life Sciences, Northeast Forestry University Lecturer: Shengnan Zhu Lecturer: Chen Li iGEMer: Zukai Liu iGEMer: Haoxian Wang Team: ZJU-China HIT-Harbin NCTU-Formosa

• Prof. Qian Wang
• Prof. Guangchao Sui
• Prof. Yuhua Li
• Prof. Chunbo Teng
• Prof. Jingying Wang

Our team

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Nanocrystal E.coli Flocculation Units N E F U NEFU