Integrated Heavy Metals Detecting Machine WearefromKoreaUniversity( - - PowerPoint PPT Presentation

Integrated Heavy Metals Detecting Machine

• We
are
from
Korea
University

(Seoul,
South
Korea)


2009
Team
:
KU_Seoul

Contents


Backgrounds Approach Experimental Procedures

1 2 3 4 5

Results Future Works

Backgrounds


Heavy
metal
pollution
became
a
social
issue
in
Korea.

?


Development Investment
in
the

 Mine
Industries


years Heavy
metal
toxicity

Heavy
Metal
Problems
in
Korea

 



Neglect
 Interest


Answer
:
HM
busters


*HM:Heavy
metal

Abandon
 Economical

 Reasons
 Solution Development
of


 HM
Busters






Continuous
contamination

Continuous
contamination
of
heavy
metals
affect

 human
public
health
problem


 

  



From
News
papers


Acid
Mine
Drainage Mutated
Frogs




Toxicity


Toxicity
of
Heavy
Metals


Approach


We
need
a
simple
and
integrated
detection
system.

Our
cell
needs
to
detect
various
metals,
 while
producing
a
simple
output.


Approach

Integrated
Heavy
Metal
detector

Brown
07
 St.Petersburg
07

Lead
detector

Case
Study


iGEM
2007

Copper
detector

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


Mission

Future
Application

‐

Capsule
based
biosensor
 AMD
 Contaminated
water
?
 Buy
‘Tylenol’
 &
grind
it!
 Mix
them
with

 a
capsule
of
the
HM
buster!
 Wait
about
1
hr
 See
color
change!


Experimental
Procedures


Experiments
Overview

Plasmid
Preparatio n


Testing
Heavy‐Metal
Detector
 
Assembly
of
Parts
 Calibration
Curve


Simple
&
Integrated
System

Preliminary
Experiments

 Module
 Color
 Intensity


Promoter Reporter
Genes
:
gpf
or
rfp
or
amd RBS Ribosome
binding
site Transcription
Factor


Heavy
metals

Heavy
metals
can
be
detected
as
fluoroscence
or
color
pigment.

Principle

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
ParsR,
Pznt
and
PyodA
originated
from
genomic
DNA
of
Escherichia
c

• li
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
]

pSB3C5

CholoramphenicolR Low/Midi‐copy


• rigin

pSB3T5


Low/Midi‐copy


• rigin

TetracyclinR

Materials

‐
Backbone
Vectors

1 2 3 4 5

← 0.5 kbp ← 1 kbp ← 2 kbp ← 3 kbp ← 4 kbp ← 6 kbp ← 5 kbp

Lane
1
:
pSB1A3‐GFP

 









2
:
pSB2K3‐RFP

 









3
:
pSB3C5‐1

 









4
:
pSB3C5‐2
 









5
:
1kb
DNA
ladder
 

Loading
DNA
:
2ul


Preliminary
Experiments


PyodA
was
selected
for
detecting
cadmium
ion.
 amd

gene
produces
aryl
acylamidase
which
converts
acetaminophen.

Parts
Assembly

Cd2+
ion
detector

PyodA amd

INS

PyodA

Cd2

PyodA

Yo dA

• plasm

PyodA

pSB3T5


am d

PyodA Low/Midi‐co py
origin TetracyclinR

(+
AAV
tag)

pSB3C5 ParsR

Pmer

CholoramphenicolR Low/Midi‐cop y
origin

pSB3C5

INS

Zn2+
and
AsO3‐
ion

Parts
Assembly

Part
I
and
PartII
are
fused
and
inserted
in
pSB3C5
plasmid

Part
I
and
PartII
are
fused
and
inserted
in
pSB3C5
plasmid

pSB3C5 ParsR

Pmer

CholoramphenicolR Low/Midi‐cop y
origin pSB3C5

INS

Parts
Assembly

Zn2+
and
AsO3‐
ion

Results


Working
of
heavy
metal
detection


pSB3T5


amd

PyodA Low/Midi‐copy


• rigin

TetracyclinR

(+
AAV
tag)

Transformation

1. Preparation


1) Cell
harvest
 2) Store
at
4℃

2.
Induction


1) Addition
of
Cd2+
to
LB 
for
60min
at
37℃
 2) Cell
harvest


3.
Detection


1) Reaction
in
Tris/HCl
(pH
9)
+
01. M
AAP
for
10min
at
37
℃
 2) Quantification
of
AP


Incubation
to
OD600~ 0.5 Escherichia
coli
DH5a

‐
Cd2+
detector


Expression
of
Red
Fluorescent
Protein



Measure
fluorescence
and
absorbance


40000 80000 120000 160000 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 Red
fluorescence Zn2+
(mM) 10000 20000 30000 40000 50000 0.2 0.4 0.6 0.8 1 Green
fluorescence AsO3‐
(mM)

Working
of
heavy
metal
detection


‐
Measurement


0.02 0.04 0.06 0.08 0.1 0.2 0.3 0.4 OD615
nm Cd2+
(mM) Expression
of

aryl
acylamidase
 Expression
of

Green
Fluorescent
Protein


Expression
of
Red
Fluorescent
Protein
 Expression
of

Green
Fluorescent
Protein


Calibrate
the
curve
based
on
concentrations Calibration
curve

‐
Calibration


y = 127878x - 97831 R² = 0.93497 40000 80000 120000 160000 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25

Red
fluorescence


Zn2+(mM)
 y = 55787x - 1896.1 R² = 0.95798 10000 20000 30000 40000 0.2 0.4 0.6 0.8 Green
fluorescence AsO3‐
(mM) y = 0.212x + 0.0042 R² = 0.9286 0.02 0.04 0.06 0.08 0.05 0.1 0.15 0.2 0.25 D615
nm Cd2+
(mM) Expression
of

aryl
acylamidase


It is possible to infer the concentration of heavy metal ion based on the calibration curve (equation)

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
Cd2+,
Zn2+,
AsO3‐
can
tell
whet her
the
water
is
drinkable,
especially
near
abandoned
mines.


Individual
parts
experiment

Preliminary
Experiments
 Assembly
of
Parts
 Calibration
Curve


Integrated
Parts
 Cd2+
,
Zn2+
,

AsO3‐

False‐color

 image
processing More
experiments Mathematics
modeling Heavy
metal
libraries

Advantages

• Integrated
system

• E.coli

can
replicate
itself


One
cell
can
detect
several
heavy
metals.
 But
what
if
there
are
more
than
several?
 We
suggest
the
Bacteria
array
 Future
Application



‐
The
limits
of
one
cell
detecting


Bacteria
Array
 Bio‐sensor
 Microarray
 ‐

Bacteria
array
:
Each
colony
detect
one
heavy
metals


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


















GREEN
Fluorescence RED
Fluorescence

Mixed
light
color
:
Red
+
Green
=
Yellow Observation
 ‐
False
color
image
processing
 Zn2+ AsO3‐

Future
Application


‐

Bacteria
array
:

Comparative
environmental
toxicology


upstream midle downstream River
1

Arsenic Mercury Lead Copper Cadmium PCBs BPH PAH Zinc

Intensity
of
fluorescence

Time
dependent Species
dependent


Example
of
data
format

River
2 River
3

Future
Application


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


Teams:
KU_Seoul
Members



• Members
(undergraduate)


– Cheol
Won
Choi
(Team
leader,
Experiment)
 – Young
Seol
Byun
(Experiment,
Wiki
 maintenance
&
Presentation)
 – Ji
Hye
Shin
&
Ji
Hui
Jang
(Experiment)
 – Cheol
Woo
Lee
(Logo
Design)
 – Simin
Kim
&
others


• Gradudates:
Hansung
Roh
(wiki
design)


































Sohyun
Kim


• Experimental
advisor:
Hyeok
Jin
Ko

• Instructor:
Prof.
In‐Geol
Choi

• Special
thanks
to
:









‐

Byung
rae
Lee(Logo
Design)



• Center
for
Teaching
and
Learning
at
Korea


University


• CSBL@KU
(http://compbio.korea.ac.kr)


Acknowledgement
(sponsors)