Tokyo-NoKoGen Championship Presentation - iGEM
Tokyo-NoKoGen Championship Presentation - iGEM
Tokyo-NoKoGen Championship Presentation - iGEM
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<strong>Tokyo</strong>-<strong>NoKoGen</strong>!<br />
@Asia Regional <strong>Championship</strong><br />
We won the mascot fun run!
<strong>Tokyo</strong>-<strong>NoKoGen</strong> 2011!<br />
Department of Biotechnology and Life Science,!<br />
<strong>Tokyo</strong> University of Agriculture and Technology!<br />
!<br />
東京農工大学 工学部 生命工学科
Problems – Heavy Metals!<br />
n Pollution<br />
Industrial waste<br />
Industrial<br />
pollution<br />
n Low amount!<br />
n High cost<br />
RECYCLE<br />
Rare metals!<br />
(Heavy metals)<br />
High-tech
E. coli collecting heavy metal ions<br />
“EcoLion”
Overview!<br />
Capture Localize & store<br />
Collect Control
For environmental consideration!<br />
Closed-system use only.<br />
What can we do to use genetically<br />
engineered E. coli for keeping the<br />
ecosystem & biodiversity.
Auto-lysis for biosafety!<br />
Inducible auto-lysis!<br />
for environmental considerations.!
Metal ion uptake!<br />
But Also they dangerous Metal are harmful ionsifare they taken to leak…<br />
E. coli in
Metal ion uptake!<br />
We need…<br />
Something that can capture metal ion<br />
So that it can localize it<br />
somewhere to store
Metal ion uptake!<br />
Capture<br />
Localize<br />
Store<br />
Metallothionein<br />
Tag<br />
BMC
Inside EcoLion<br />
Endogenous<br />
transporter<br />
Cd 2+<br />
Capture<br />
Metallothionein<br />
Tag<br />
BMC<br />
Localize<br />
&<br />
Store
Metallothionein<br />
N-‐terminus needed<br />
for efficient packaging<br />
Cadmium(II) binding metallothioneins<br />
SmtA<br />
fMT<br />
PduP 1-‐18<br />
<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011<br />
BBa_K519010<br />
From Synechococcus sp. PCC7942<br />
Groningen 2009<br />
From Fucus Vesiculosus<br />
<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011<br />
BBa_K519012<br />
N-‐terminus of propionaldehyde<br />
dehydrogenase enzyme in<br />
pduBMC.<br />
Protein tag to localize into BMC
Evaluated constructs<br />
1.) SmtA<br />
P const.(Low)<br />
RBS pduP 1-‐18 -‐smtA<br />
Term.<br />
Term<br />
.<br />
BBa_K519015 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
2.) fMT<br />
P const.(Low)<br />
P const.(Low)<br />
RBS smtA<br />
TermTerm.<br />
.<br />
BBa_K519018 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
P const.(High)<br />
RBS smtA<br />
TermTerm.<br />
.<br />
BBa_K519022 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
RBS pduP 1-‐18 -‐fMT<br />
BBa_K519014 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
Term. Ter<br />
m.
Hypothesis<br />
Cd2+ low concentraXon<br />
High<br />
WT WT WT<br />
E. coli expressing metallothionein will tolerate<br />
high Cd 2+ concentraXon.<br />
WT
Hypothesis<br />
Cd 2+<br />
Metallothionein will bind with Cd 2+ and<br />
prevent cytotoxicity.<br />
Metallothionein
Result:<br />
OD 595 595<br />
0.5<br />
0.4<br />
0.3<br />
0.2 0.2<br />
0.1<br />
0.1<br />
0<br />
0<br />
P const.(Low)<br />
Cd2+ Cd 0 µM medium<br />
2+ 200 µM medium<br />
0 1 2 3 4 5 6 7 8 9<br />
0 1 2 3 4 5 6 7 8 9<br />
RBS smtA<br />
SmtA<br />
Term.<br />
Term.<br />
Bba_K519018 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
Time (hour)<br />
Time (hour)<br />
WT<br />
SmtA<br />
WT<br />
SmtA expressing E. coli tolerates<br />
high Cd 2+ concentraXon.
Result: PduP1-18 fused SmtA and fMT<br />
OD 595<br />
595<br />
0.35 0.3<br />
0.25 0.3<br />
0.25<br />
0.2<br />
0.2<br />
0.15<br />
0.15<br />
0.1<br />
0.05<br />
0<br />
P const.(Low)<br />
Cd2+ Cd 0µM medium<br />
2+ 180 µM medium<br />
0 1 2 3 4 5 6 7 8<br />
RBS PduP 1-‐18 SmtA<br />
RBS PduP 1-‐18 fMT<br />
Term.<br />
Bba_K519014, K519015 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
Time (hour)<br />
WT fMT<br />
SmtA fMT<br />
WT<br />
SmtA<br />
E. coli expressing PduP 1-‐18 fused fMT<br />
tolerates high Cd 2+ concentraXon.
Result: Changing promoter<br />
OD 595<br />
0.3<br />
0.3<br />
0.25<br />
0.25<br />
0.2<br />
0.2<br />
0.15<br />
0.15<br />
0.1<br />
0.1<br />
0.05<br />
0.05<br />
0<br />
Cd2+ Cd 300 µM medium<br />
2+ 300 µM medium<br />
0 2 4 6 8<br />
0 2 4 6 8<br />
Pconst.(High)-‐SmtA<br />
Pconst.(High)-‐PduP 1-‐18 fMT<br />
Pconst.(Low)-‐SmtA<br />
Pconst.(Low)-‐SmtA<br />
Pconst.(Low)-‐PduPfMT<br />
Pconst.(Low)-‐PduP<br />
WT<br />
1-‐18fMT WT<br />
Higher expression of metallothionein with Pconst.(High)<br />
Higher tolerance for Cd2+ Time Time (hours) (hours)
Result: Changing promoter<br />
OD 595 at 7.5h<br />
0.25<br />
0.2<br />
0.15<br />
0.1<br />
0.05<br />
0<br />
Difference in OD 595 of E. coli at<br />
different Cd 2+ concentraXons<br />
0 200 400 600 800 1000<br />
Cd 2+ conentraXon (µM)<br />
WT<br />
E. coli expressing and<br />
can tolerate high Cd2+ SmtA<br />
concentraXon.<br />
Pconst.(High)-‐PduP 1-‐18 fMT<br />
PduP 1-‐18<br />
fMT<br />
Pconst.(High)-‐SmtA
Metallothionein<br />
E. coli expressing<br />
SmtA PduP 1-‐18 -‐fMT<br />
can tolerate Cd 2+ containing medium<br />
Metallothionein is capturing<br />
Cd 2+ inside the cell.
Bacterial Microcompartment<br />
So…<br />
What is BMC?
PduBMC<br />
PduBMC<br />
Citrobacter freundii<br />
100 nm<br />
Propanediol-‐uPlizing<br />
Bacterial Microcompartment<br />
Protein shell<br />
in bacteria<br />
for processing<br />
enzymaPc reacPons
Localization of pduP 1-18 -GFP<br />
1. pET30c-‐P h -‐pduP 1-‐18 -‐GFP (T7promoter (-‐))<br />
P const.(H)<br />
RBS pduP 1-‐18 -‐GFP<br />
Term<br />
.<br />
Term<br />
.<br />
2. pET30c-‐pduABJKNU-‐P h -‐pduP 1-‐18 -‐GFP (T7promoter (+))<br />
P const.(H)<br />
RBS pduA RBS pduB RBS pduJ RBS pduK RBS pduN RBS pduU RBS pduP 1-‐18 -‐GFP<br />
BBa_K519001 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
1. Control (without BMC)<br />
2. Microscopic images pduP 1-‐18 -‐GFP inside E. coli<br />
pduP 1-18 -GFP localized in pduBMC?
How can we “collect” EcoLion?<br />
How will it be collected?
Control & collection of EcoLion<br />
Control→phototaxis<br />
Collection→aggregation
How does Phototaxis work?!<br />
Smooth<br />
swimming<br />
under dark<br />
under dark<br />
tumbling
Result: Phototaxis<br />
Under the light<br />
Size of colony will not show much change.<br />
tumbling
Result: Phototaxis<br />
In the dark<br />
Size of colony will become bigger.<br />
Smooth<br />
swimming
Result: Phototaxis<br />
Relative colony size (%)<br />
BBa_K317028 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2010)<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Light<br />
control(Light) control(Dark)<br />
Light!<br />
control<br />
Dark<br />
Dark<br />
+Phototaxis device<br />
photo(Light) photo(Dark)
How does Antigen43 work?!
How does Antigen43 work?!
Result: Aggregation<br />
aggregation!<br />
& !<br />
precipitation!<br />
Upper’s optical density !<br />
will decrease.
Result: Aggregation<br />
OD 595<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
P LlacO-‐1<br />
RBS<br />
AnXgen43 (+IPTG)<br />
An?gen 43<br />
0 1 2 3<br />
Time with standing (h)<br />
Term.<br />
BBa_K519021 (<strong>Tokyo</strong>-‐<strong>NoKoGen</strong> 2011)<br />
Control (-‐IPTG)<br />
Control (+IPTG)<br />
AnXgen43 (-‐IPTG)<br />
Term.<br />
AnXgen43 control<br />
(+IPTG) (-‐IPTG) (+IPTG) (-‐IPTG)
Control & collection by light signals
Problem...!<br />
Environmental considerations<br />
We should keep EcoLion under control.
EcoLion dies under red light.!<br />
Working in the dark. Lysis under light.
Lysis: Breaking down periplasm!<br />
E. coli<br />
cell<br />
Peptidoglycan<br />
Cytosol<br />
Endolysin<br />
Holin<br />
Glycosylase !<br />
reaction<br />
Outer membrane<br />
Inner membrane<br />
Antiholin<br />
Periplasm
Lysis: Results!<br />
OD 660<br />
3!<br />
2.5!<br />
2!<br />
1.5!<br />
1!<br />
0.5!<br />
IPTG induction<br />
0!<br />
0! 2! 4! 6! 8! 10! 12! 14!<br />
Time (hour)<br />
(+) IPTG!<br />
(-) IPTG!
Lysis: Results!<br />
Colony forming unit / mL<br />
2.5x10 9<br />
2.5E+09!<br />
2.0x10 9<br />
2.0E+09!<br />
1.5E+09!<br />
1.5x10 9<br />
1.0E+09!<br />
1.0x10 9<br />
5.0E+08!<br />
5.0x10 8<br />
0.0E+00! 0<br />
CFU / mL!<br />
of E. coli with Lysis genes<br />
Lysis is correctly<br />
being induced<br />
without IPTG! With IPTG!<br />
Without IPTG ! ! With IPTG
Solution!<br />
Environmental considerations<br />
We can keep EcoLion under control !!
Summary!<br />
Capture<br />
Metallothionein<br />
Localization<br />
BMC<br />
Heavy metal ions<br />
contaminated<br />
water
Summary!
Summary!
Summary!<br />
Control<br />
Phototaxis
Summary!
Summary!
Summary!<br />
Collection<br />
Aggregation
Summary!<br />
We can collect HEAVY<br />
METALS easily!!
Summary!
Summary!<br />
Lysis<br />
Safe!!
Perspective!<br />
n Clean up heavy metal<br />
wastes & pollutions<br />
Industrial waste<br />
Industrial<br />
pollution<br />
n Recycle heavy metals<br />
RECYCLE<br />
Rare metals!<br />
(Heavy metals)
Achievement!<br />
Aiming at Parts & Device Submitted Work<br />
Capture Metallothionein<br />
SmtA 3<br />
fMT 1<br />
Localize BMC 6<br />
Photo control<br />
Phototaxis 0<br />
Light actuators 1<br />
Sedimentation Aggregation 1<br />
Biosafety Lysis 1<br />
Uptake Metal transporter 2<br />
✔<br />
✔<br />
Improved an existing fMT.!<br />
Improved an exsiting lysis device.!<br />
"<br />
✔<br />
✔<br />
✔<br />
✔<br />
✔
Acknowledgment!<br />
Department of Biotechnology and Life Science,"<br />
<strong>Tokyo</strong> University of Agriculture and Technology"<br />
"<br />
Ultizyme International, Ltd.
Nasa SAVORY, Kotone MIYAKE, Shoko SAITO, Saki NAKASHIMA, Yuki<br />
UCHIKURA, Chifuku MITA, Masataka ARAKI, Koichi SUMIDA, Eri KAMIO,<br />
Hikaru SEKIGUCHI, Mitsuharu NAKAJIMA, Kaori TSUKAKOSHI, Assist.<br />
Prof. Stefano FERRI, Prof. Kazunori IKEBUKURO, and Prof. Koji SODE.!
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