integrated heavy metals detecting machine
play

2009Team:KU_Seoul Integrated Heavy Metals Detecting Machine WearefromKoreaUniversity( Seoul,SouthKorea ) Contents 1 Backgrounds 2 Approach 3 Experimental Procedures 4 Results 5 Future Works

0 downloads 7 Views 2,39 MB Size Report
  1. 2009
Team
:
KU_Seoul Integrated Heavy Metals Detecting Machine We
are
from
Korea
University

( Seoul,
South
Korea )
 •

  2. Contents 
 1 Backgrounds 2 Approach 3 Experimental Procedures 4 Results 5 Future Works

  3. Backgrounds


  4. 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.

  5. 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


  6. Approach


  7. 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.

  8. Mission We
chose
three
heavy
metals,
Zinc,
Arsenic
and
Cadmium,
as
our
target.


  9. 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


  10. Experimental
Procedures


  11. Experiments
Overview Preliminary
Experiments

 Plasmid
Preparatio n
 
Assembly
of
Parts
 Testing
Heavy‐Metal
Detector
 Calibration
Curve
 Module
 Color
 Intensity
 Simple
&
Integrated
System

  12. 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.

  13. 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
]

  14. Materials ‐
Backbone
Vectors Low/Midi‐copy
 Tetracyclin R origin pSB3T5
 pSB3C5 Low/Midi‐copy
 Choloramphenicol R origin

  15. 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

  16. 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.

  17. 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

  18. 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

  19. Results


  20. 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


  21. 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


  22. 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

  23. 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


  24. Future
Applications


  25. 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


  26. Advantages • 
 Integrated
system 
 • E.coli

 can
replicate
itself


  27. 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


  28. Future
Application
 ‐
 
Bacteria
array
:
Each
colony
detect
one
heavy
metals
 Bio‐sensor
 Bacteria
Array
 Microarray


  29. Future
Application
 ‐

Bacteria
array

:
How
does
it
work?
 RFP
based
 E.
coli GFP
based
 E.
coli False
color
image

 processing Histidine‐Tag
On
flagellar


  30. Future
Application
 ‐
 False
color
image
processing
 Zn 2+ RED
Fluorescence 















 GREEN
Fluorescence AsO 3‐ Observation
 Mixed
light
color
 :
 Red 
+
 Green
=
Yellow

  31. 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

  32. 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 Documents


metals lead cadmium and mercury work schedule for heavy
Metals (lead, cadmium and mercury)

HELCOM CORE INDICATOR Metals (lead, cadmium and mercury) Work schedule for

six classes 6 some heavy metals
Six Classes 6. Some Heavy

Six Classes 6. Some Heavy Metals Dr. Graham F. Peaslee

use of nanotechnology in remediation of radionuclides and
Use of Nanotechnology in Remediation of

Use of Nanotechnology in Remediation of Radionuclides and Heavy Metals Frank

feasibility of fungi to remove heavy metals from mine
Feasibility of Fungi to Remove Heavy

Feasibility of Fungi to Remove Heavy Metals from Mine Wastewater CENE

on heavy metals
ON HEAVY METALS? Robert van de Graaff,

ARE WE SOMETIMES TOO HEAVY ON HEAVY METALS? Robert van de Graaff, PhD van de

presentation outline
Presentation Outline Traditional

2/22/2012 Presentation Outline Traditional Engineering Solutions The

impact of airborne heavy metals on lung impact of
Impact of Airborne Heavy Metals on Lung

https://www.uab.edu/medicine/src/ Impact of Airborne Heavy Metals on Lung

interplay of incipient magnetism and superconductivity in
Interplay of incipient magnetism and

Interplay of incipient magnetism and superconductivity in heavy-fermion metals

development of in field portable sensor for detection of
Development of In-field Portable

Development of In-field Portable Sensor for Detection of Phosphates and Heavy

determination of heavy metals in canned dry milk and fish
Determination of Heavy Metals in

Determination of Heavy Metals in Canned Dry-Milk and Fish from Supermarkets

boot camp transfusion reactions
Boot Camp Transfusion Reactions Dr.

Boot Camp Transfusion Reactions Dr. Kristine Roland Regional Medical Lead

lnterference of hepatotoxicity with endocrine activity in
lnterference of hepatotoxicity with

SETAC Rome, May 2018 lnterference of hepatotoxicity with endocrine activity

detection and elimination
Detection and Elimination Todd Borci

Stormwater Illicit Discharge Detection and Elimination Todd Borci EPA Region

newton medical center
NEWTON MEDICAL CENTER Position

NEWTON MEDICAL CENTER Position Statement on Pain Management and Opioid Usage

niel dalal
Niel Dalal MPH 2013 Gastroschisis is a

Niel Dalal MPH 2013 Gastroschisis is a congenital fissure in the abdominal

medi meal
MEDI-MEAL A Reference Guide to

MEDI-MEAL: A Reference Guide to Over the Counter Drugs // Introduction 1/37

a
a hi Oa cD 0 n.j U, to N t cJ

r n.j Lt} N >z tO (U a hi Oa cD 0 n.j U, to N t cJ C tO

pharmacist role in continuous glucose monitoring cgm
Pharmacist Role in Continuous Glucose

5/30/18 Pharmacist Role in Continuous Glucose Monitoring (CGM) Gabriella

pl ayi ng i n pl ast e r t he basi cs of spl i nt i ng
PL AYI NG I N PL AST E R T HE

PL AYI NG I N PL AST E R T HE BASI CS OF SPL I NT I NG K a ri

2013 ulm student research symposium platform oral
2013 ULM Student Research Symposium

2013 ULM Student Research Symposium PLATFORM (ORAL) PRESENTATIONS College of

as seasons change so do we age
As seasons change, so do we age!

As seasons change, so do we age! Aging Adults Aging Statistics Persons 65

city of los angeles
City of Los Angeles 2018 Q3

City of Los Angeles 2018 Q3 Utilization Update Presented By: Justin Cao, MPH