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

2014 iGEM 1 NEFU-China Northeast Forestry University

2 N orth E ast F orestry U niversity NEFU N E F U Nanocrystal E.coli Flocculation Units

Inspiration 3 Shocking News ： Shocking News ： - Two million people at risk of death from poisoned water The water in northeastern Indian state of 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. http://news.163.com/13/0731/08/953M00U80001124J_all.html

Inspiration 4 Cadmium Pollution – A Serious Concern

Inspiration 5 Cadmium Pollution – A Serious Concern Soil cadmium contents in 30 provinces of China 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 Cd/mg ． kg -1 China environmental quality standard for soil GB15618-1995

Inspiration 6 Visiting Sewage Plant Inductively Coupled Plasma - Optical Emission Spectrometry China environmental quality standard for water GB8978-1996

Sources 7 Ni-Cd Batteries Non-ferrous Industrial Waste metal smelting Automobile Exhaustion Pesticides & Fertilizers

Diseases 8 Cancer Diabetes Itai-Itai Disease Pneumonia Anemia

SURVEY 9 Ethical issues  Which is the best way to deal with heavy metal pollution?  What kind of problems will be triggered by biological treatment? 185 respondents (93 male, 92 female)

Our Design 10 10 Module 1 Module 2 Module 3 Detecting Recycling Flocculating Detect the Synthesize Collect the presence of the CdS nanocrystals Cadmium ions nanocrystals by flocculating

Which is suitable in our Project? 11 11 Metal System Members Host sensed 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 …… Pennella, M. A. Giedroc Biometals, 2005. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

12 12 Stop smtB smtO-P smtA Stop S.D. -10(smtB) Inverted repeat smt B smt A -10(smtA) Inverted repeat S.D. Synechococcus PCC7942 smt -locus structure Huckle, J. W. Mol Microbiol, 1993. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

13 13 Cd 2+ Stop smtB smtO-P smtA Stop Huckle, J. W. Mol Microbiol, 1993. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

14 14 pACYC184 Cd 2+ MCS amilCP Stop smtB smtO-P smtA Stop Huckle, J. W. Mol Microbiol, 1993. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Results 15 15 smt -locus (+) strain: more tolerant of Cd 2+ 1.4 smtB -OP- smtA (+) 1.2 Control 1.0 0.8 OD 540 0.6 0.4 0.2 0.0 0 20 40 60 80 100 120 140 Time (h) Growth of cells containing smt - locus element in LB medium supplemented with 2μM Cd 2+ DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Results 16 16 More sensitive to Cd 2+ with shorter period than Zn 2+ Cd 2+ 2.0 Zn 2+ 3.0 2.5 1.5 2.0 OD 450 1.0 OD 450 1.5 Cd 2+ 1.0 0.5 Zn 2+ 0.5 0.0 0.0 2 5 10 20 50 0 2 4 6 8 10 12 Time (h) Concentration (μM ) Cd 2+ and Zn 2+ (1-5 0μM) suppl ement for 2h Cd 2+ and Zn 2+ (2μM) s upplement for 1-12h Metal-induced expression of the pigments DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Results 17 17 Visualized Output 500 (μM) 200 (μM) (μM) 100 (μM) 50 (μM) 20 (μM) 10 Saturation DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Equations 18 18 Cd 2+ ① dp = r 1 (P - p) Cd 2+ Cd 2+ ② dX/dt = x 0 (1-f(p)) ③ dY/dt = y 0 (1-X/x 0 t) Stop smtB smtO-P amilCP Stop DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Equations 19 19 (4) Y= y 0 tf(P – e tr1 ) Y ---- Expression level of reporter gene y 0 ---- The maximum amount per unit time P ---- Concentration of Cd 2+ in extracellular p ---- Concentration of Cd 2+ in cell r 1 ---- The moving rate that Cd 2+ enter into cells Simplifying the equation (4), we work out the equation (5). (5) Y= y 0 tf(P) DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Data 20 20 2.0 1.5 OD 600 1.0 0.5 Reporter expression 0.0 0.0 0.5 1.0 1.5 2.0 Time (h) Fig.1 Cdmium-induced expression of the Fig.2 Cdmium-induced expression of the pigment (amilCP) with a constant pigment (amilCP) with different concentration (1μM) concentrations DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

21 21 3.0 2.0 2.5 OD 600 Fit 1 1.5 Y = 1.021 - 0.0003145P 2 + 0.04481P RMSE=0.3657 1.0 R 2 =0.8739 Fit 2 0.5 Y = 3.063 – 2.321P -0.2802 Reporter expression Fit 1 RMSE=0.0589 Fit 2 R 2 =0.9967 0.0 0 20 40 60 80 100 120 Concentration (μM) Fig.3 DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Our Design 22 22 Module 1 Module 2 Module 3 Detecting Recycling Flocculating Detect the Synthesize Collect the presence of the CdS nanocrystals Cadmium ions nanocrystals by flocculating

CdCl 2 + Na 2 S CdS 23 23 CdS + CdS Nano Short Peptide pET28b MCS S 2- Cd 2+ Short Peptide CdS Nano CP25 CDS7 Stop 0.2μm Jensen, P.R. Appl Environ Microbiol, 1998. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

CdCl 2 + Na 2 S CdS 24 24 CdS + CdS Nano Short Peptide pET28b MCS S 2- Cd 2+ Short Peptide CdS Nano CP25 CDS7 Stop Jensen, P.R. Appl Environ Microbiol, 1998. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

25 25 pET28b MCS S 2- Cd 2+ Short Peptide CdS Nano CP25 CDS7 Stop 0.2μm One E.coli cell (TEM) Jensen, P.R. Appl Environ Microbiol, 1998. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Our Design 26 26 Module 1 Module 2 Module 3 Detecting Recycling Flocculating Detect the Synthesize Collect the presence of the CdS nanocrystals Cadmium ions nanocrystals by flocculating

27 27 pET28b MCS IPTG T7 promoter Flocculation gene Stop Fang Ma, China water and wastewater, 2003. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

28 28 pET28b MCS IPTG T7 promoter Flocculation gene Stop Fang Ma, China water and wastewater, 2003. DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Results 29 29 Improved sediment ability 100 Floc. （ + ） Floc. （ - ） 80 Settling Cells (%) 60 40 20 0 0 5 10 15 20 Settling Time (min) DETECTING RECYCLING FLOCCULATING MODELING SYSTEM SYSTEM SYSTEM

Summary 30 30 Module 1 Module 2 Module 3 Detecting Recycling Flocculating Detect the Synthesize Collect the presence of the CdS nanocrystals Cadmium ions nanocrystals by flocculating 0.2μm

Achievement 31 31 • Parts We submitted four functional parts: BBa_K1509000 BBa_K1509001 BBa_K1509002 BBa_K1509003 • Collaboration Team ZJU-China Team HIT-Harbin • Human practice Conferences Online survey Visiting sewage plant

Human Practice 32 32

Acknowledgement 33 33 Prof. Qian Wang Lecturer: Shengnan Zhu Prof. Guangchao Sui Lecturer: Chen Li Prof. Yuhua Li iGEMer: Zukai Liu Prof. Chunbo Teng iGEMer: Haoxian Wang Prof. Jingying Wang Team: ZJU-China HIT-Harbin NCTU-Formosa 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

Our team 34 34

NEFU 35 35 N E F U Nanocrystal E.coli Flocculation Units