2009 Team : KU_Seoul Integrated Heavy Metals Detecting Machine We are from Korea University ( Seoul, South Korea ) •
Contents 1 Backgrounds 2 Approach 3 Experimental Procedures 4 Results 5 Future Works
Backgrounds
Heavy Metal Problems in Korea ? Interest Heavy metal toxicity Answer : HM busters Neglect *HM:Heavy metal years Development Abandon Solution Investment in the Economical Development of Mine Industries Reasons HM Busters Heavy metal pollution became a social issue in Korea.
Toxicity of Heavy Metals Acid Mine Drainage Mutated Frogs From News papers Continuous contamination Toxicity Continuous contamination of heavy metals affect human public health problem
Approach
Approach Our cell needs to detect various metals, while producing a simple output. Case Study iGEM 2007 Brown 07 St.Petersburg 07 Lead detector Copper detector Integrated Heavy Metal detector We need a simple and integrated detection system.
Mission We chose three heavy metals, Zinc, Arsenic and Cadmium, as our target.
Future Application ‐ Capsule based biosensor AMD Buy ‘Tylenol’ Contaminated water ? & grind it! Mix them with a capsule of the HM buster! See color change! Wait about 1 hr
Experimental Procedures
Experiments Overview Preliminary Experiments Plasmid Preparatio n Assembly of Parts Testing Heavy‐Metal Detector Calibration Curve Module Color Intensity Simple & Integrated System
Principle Transcription Factor Heavy metals RBS Promoter Ribosome binding site Reporter Genes : gpf or rfp or amd Heavy metals can be detected as fluoroscence or color pigment.
Materials Backbone Plasmids Plasmid pSB3C5 with part BBa_J04450 : 2009 Kit Plate 1 [5C] Plasmid pSB3T5 with part BBa_J04450 : 2009 Kit Plate 1 [9C] Promoters Promoter P arsR , P znt and P yodA originated from genomic DNA of Escherichia c oli XL‐1 Blue Protein Coding Sequences Green fluorescent protein [BBa_E0044] : 2009 Kit Plate 1 [14G] Red fluorescent protein [BBa_E1010] : 2009 Kit Plate 1 [18F] Aryl acylamidase protein : New biological part [Part:BBa_K271000] [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleot ide&list_uids=227462445&dopt=GenBank ]
Materials ‐ Backbone Vectors Low/Midi‐copy Tetracyclin R origin pSB3T5 pSB3C5 Low/Midi‐copy Choloramphenicol R origin
Preliminary Experiments 1 2 3 4 Lane 1 : pSB1A3‐GFP 5 2 : pSB2K3‐RFP 3 : pSB3C5‐1 4 : pSB3C5‐2 5 : 1kb DNA ladder ← 6 kbp ← 5 kbp ← 4 kbp ← 3 kbp ← 2 kbp Loading DNA : 2ul ← 1 kbp ← 0.5 kbp
Parts Assembly Cd 2+ ion detector am P yodA d (+ AAV tag) Tetracyclin R pSB3T5 Low/Midi‐co Amd_R P yodA _R py origin A A P yodA amd V P yodA _F Amd_F Cd 2 Metal‐responsive promoter (extended spacer region) pSB3T5_R + activation No expression INS yodA P yodA yodA P yodA Yo pSB3T5_F Positioned for binding Cd2+ pSB3T5 dA oplasm before it could enter the cyt P yodA P yodA was selected for detecting cadmium ion. amd gene produces aryl acylamidase which converts acetaminophen.
Parts Assembly Zn 2+ and AsO 3‐ ion rfp P mer gfp Low/Midi‐cop y origin pSB3C5 P arsR Choloramphenicol R (As 3+ ) 2 ArsR + ArsR arsR P arsR Derepression INS (ArsR) 2 Repression arsR P arsR (ArsR) 2 pSB3C5 gfp P arsR Part I and PartII are fused and inserted in pSB3C5 plasmid
Parts Assembly Zn 2+ and AsO 3‐ ion rfp P mer gfp Low/Midi‐cop y origin pSB3C5 P arsR Choloramphenicol R Hg 2+ Derepr Zn ession Repr essio Me n rR merRTPC merRTPCD P znt P mer DAB AB INS MerTPCDAB pro Hg 2+ → H teins g 0 pSB3C5 Transpo rtation P mer Part I and PartII are fused and inserted in pSB3C5 plasmid
Results
Working of heavy metal detection ‐ Cd 2+ detector 1. Preparation 1) Cell harvest Store at 4 ℃ Escherichia coli DH5a 2) Incubation to OD 600 ~ 0.5 Transformation 2. Induction 1) Addition of Cd 2+ to LB for 60min at 37 ℃ P yodA amd 2) Cell harvest (+ AAV tag) Tetracyclin R pSB3T5 3. Detection 1) Reaction in Tris/HCl (pH 9) + 01. Low/Midi‐copy M AAP for 10min at 37 ℃ origin 2) Quantification of AP
Working of heavy metal detection ‐ Measurement Expression of Red Fluorescent Protein Expression of Green Fluorescent Protein Expression of aryl acylamidase 0.08 50000 160000 40000 Green fluorescence 0.06 120000 Red fluorescence OD 615 nm 30000 0.04 80000 20000 40000 0.02 10000 0 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 Zn 2+ (mM) AsO 3‐ (mM) Cd 2+ (mM) Measure fluorescence and absorbance
Calibration curve ‐ Calibration It is possible to infer the concentration of heavy metal ion based on the calibration curve (equation) Expression of Red Fluorescent Protein Expression of Green Fluorescent Protein Expression of aryl acylamidase 40000 0.08 160000 y = 127878x - 97831 y = 0.212x + 0.0042 R² = 0.93497 Green fluorescence 30000 R² = 0.9286 0.06 120000 D615 nm Red fluorescence y = 55787x - 1896.1 20000 0.04 80000 R² = 0.95798 10000 0.02 40000 0 0 0 0 0.2 0.4 0.6 0.8 0 0.05 0.1 0.15 0.2 0.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 AsO 3‐ (mM) Zn 2+ (mM) Cd2+ (mM) Calibrate the curve based on concentrations
Experiment Summary • We successfully constructed metal detection circuits in E. coli. 1. Zinc detector using RFP as a reporter was constructed and worked at the range of 1~2mM concentration 2. Arsenic detector using GFP as a reporter was constructed and worked at the range of 0.15~1mM concentration 3. Cadmium detector using AMD as a reporter was constructed and worked at the range of 0.2~0.4mM concentration
Future Applications
Future Works Biological detection system comprising Cd 2+ , Zn 2+ , AsO 3‐ can tell whet her the water is drinkable, especially near abandoned mines. Heavy metal libraries False‐color image processing More experiments Mathematics modeling Integrated Parts Cd 2+ , Zn 2+ , AsO 3‐ Individual parts experiment Preliminary Experiments Assembly of Parts Calibration Curve
Advantages • Integrated system • E.coli can replicate itself
Future Application ‐ The limits of one cell detecting One cell can detect several heavy metals. But what if there are more than several? We suggest the Bacteria array
Future Application ‐ Bacteria array : Each colony detect one heavy metals Bio‐sensor Bacteria Array Microarray
Future Application ‐ Bacteria array : How does it work? RFP based E. coli GFP based E. coli False color image processing Histidine‐Tag On flagellar
Future Application ‐ False color image processing Zn 2+ RED Fluorescence GREEN Fluorescence AsO 3‐ Observation Mixed light color : Red + Green = Yellow
Future Application ‐ Bacteria array : Comparative environmental toxicology Species dependent Time dependent River 1 River 2 River 3 upstream midle downstream Arsenic Mercury Lead Copper Cadmium PCBs BPH PAH Zinc Example of data format Intensity of fluorescence
Team : KU_Seoul Korea University is located in Seoul, Kor ea We started from 2008 Fall Microbiology Class 3 instructors, 3 advisors (graduates), 10 UGs Korea University Latitude 37°35'8.11"N Longitude 127° 1'35.10"E
Recommend
More recommend
