Integrated Heavy Metals Detecting Machine WearefromKoreaUniversity( - PowerPoint PPT Presentation

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