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Lysis ~E.coli collecting heavy metal ions in BMC~
Tokyo-NoKoGen
We developed the , for the collection of
heavy metals from the environment, aimed at
cleaning up heavy-metal pollution as well as for the
mining of valuable metals for industrial applications.
A bacterial micro-compartment (BMC) was
engineered into our E. coli micromachine to act as a
tank to accumulate heavy metals. Heavy metal ions,
such as Cadmium (II), that were taken up by the cell
will be trapped inside the BMC by a metallothionein
fused to a BMC-localizing tag. Using BMC in such a
way will be very advantageous as it may achieve the
accumulation of high concentrations of heavy metals
in one place. The EcoLion that has stored heavy
metals will be conveniently collected by light control
using phototaxis or self-aggregation. In addition, we
constructed a lysis device for biosafety. The lysis
device can automatically kill any cells they are
released in the environment.
Students: Nasa SAVORY, Chifuku MITA, Eri KAMIO, Koichi SUMIDA, Kotone MIYAKE, Masataka ARAKI, Shoko SAITO, Saki NAKASHIMA, Yuki UCHIKURA, and Hikaru SEKIGUCHI Advisors: Kaori TSUKAKOSHI, and Mitsuharu NAKAJIMA Instructors: Assist. Prof. Stefano FERRI, Prof. Kazunori IKEBUKURO, and Prof. Koji SODE Tokyo University of Agriculture & Technology E-mail: [email protected]
Lysis for biosafety
0
0.5
1
1.5
2
2.5
3
0 5 10 15
OD
66
0
(hour)
(+) IPTG
(-) IPTG
IPTG induction
0
1
2
3
4
5
0 5 10 15 20 25
OD
66
0
(hour)
pSB1C3-RFP (control)
Lysis system withoutantiholin
Lysis system withantiholin
E. coli
cell
Peptidoglycan
Cytosol
Endolysin
Holin
Outer membrane
Inner
membrane
Periplasm Glycosylase
reaction
Antiholin
Our NEW part BBa_K519020
Within 2 hours after addition of IPTG, the OD660 went down by
80%, indicating that the lysis genes were successfully
induced by IPTG.
The inducible lysis system consisting of endolysin and
holin functioned the same way regardless of antiholn.
(3) Phototaxis allows controlling cells.
(blue circles show initial sizes of colonies)
NpSRll (retinylidene photoreceptors sensory rhodopsin) transmits signals
to its transducer protein, NpHtrll, which has domains that are homologous
to chemotaxis transducers of StTar.
Under Light Under Dark
Smooth
swimming Tumbling
0
20
40
60
80
100
120
Are
a (%
)
0
20
40
60
80
100
120
Are
a (%
)
Evaluation of phototaxis by measuring change of colony size in 48 hour.
(4) Cells aggregate for sedimentation.
0
0.2
0.4
0.6
0.8
1
0 1 2 3
OD
59
5
(hour)
pSB1C3-PLlacO-1-RBS-Antigen43-DT(+ IPTG)
pSB1C3-PLlacO-1-RBS-Antigen43-DT(non IPTG)
Control(+ IPTG)
Control(non IPTG)
Appearance of E. coli sinking (3 hour from standing)
Evaluation of aggregation by measuring OD595 of the culture
1 cm below the surface
E. coli expressing Antigen43 showed a significant decrease in OD595 compared to
the E.coli without Antigen43 gene as we expected. Antigen43 can be used to
aggregate cells for sedimentation.
Inner
membrane
Periplasm
Outer
membrane
Model of the biogenesis and processing of the aggregation protein Antigen43.
Llaco-1
(1) Metallothioneins capture Cd2+.
Our NEW part BBa_K519010
SmtA :
Pconst.
RBS smtA
Term. Term.
Metallothionein cloned
from Synechococcus sp. PCC7942
Metallothionein from Fucus vesiculosus
(Groningen 2009)
RBS pduP1-18-fMT
Pconst. Term. Term.
fMT :
(PduP1-18 :Protein tag to transport into BMC)
Our NEW part BBa_K519013
PduA, -B, -B’, -J, -K, -N, -T, -U : PduBMC shell proteins
PduP1-18: Localization-tag peptide for packaging proteins into the PduBMC.
Confirmation of PduBMC expression and
PduP1-18-GFP localization into BMC.
We focused on a propanediol-utilizing
BMC (PduBMC) derived from Citrobacter
freundii to localize and concentrate heavy
metals captured by metallothioneins.
BMCs are internal compartments, which
certain bacteria naturally have and use to
optimize metabolic reactions as natural
bioreactors.
(2) BMC for localization of Cd2+.
Our NEW part BBa_K519001
We could not confirm localization
of PduP1-18-GFP in PduBMC with certainty.
The fluorescence
microscopic image
of E. coli cells co-
expressing GFP
with pduP tag and
PduBMC
This image looked similar to a control without BMC shell genes.
The lysis device coding Holin and Endolysin causes auto-lysis of E. coli cells. Holin
proteins cause “pores” in the inner membrane of E.coli,which allows endolysin to
access and break down the peptidoglycan in the periplasm.
Our NEW part BBa_K519021
If we regulate genetically engineered E. coli, such as EcoLion,
using a red light actuator (BBa_K519030) for inducing lysis,
then those E. coli can work in the environment at minimum
effects for the ecosystem and also at low risk of biohazard.
What can we do to use EcoLion in the environment? – Genetically
engineered E. coli cells should not be freely distributed to the environment for keeping
the ecosystem and biological diversity and preventing unexpected effects, so we came
up with the idea of Inducible-lysis device to destroy the E. coli after finishing its work.
Phototaxis
Capture
Localization
Aggregation
Uptake
Heavy metal ions
BBa_K317028 (Tokyo-NoKoGen 2010)
BBa_K317040 (Tokyo-NoKoGen 2010)
+ Phototaxis device Control
LIG
HT
LIG
HT DA
RK
DA
RK
BMC + BMC - The fluorescence
microscopic image
of E. coli cells
expressing GFP
with pduP tag as a
control
Abstract
We constructed 14 new BioBricks and
demonstrated that four devices
(Metallothionein, Phototaxis,
Aggregation, and Lysis) successfully
functioned as expected. The EcoLion
composed of those BioBricks
ultimately can be applied for collecting
a number of different toxic or valuable
molecules, not only heavy metals, by
using specific target-binding peptides
or proteins. In addition, our lysis
device may eventually allow us to use
genetically engineered E. coli in the
environment keeping the ecosystem
and biodiversity.
Conclusion
0
0.05
0.1
0.15
0.2
0.25
0 200 400 600 800 1000
OD
595
at 7
.5h
[Cd2+] (mM)
WT
Pconst(H)-SmtA
Pconst(H)-PduP1~18-fMT
Pconst.(H)-PduP1~18-fMT
Our NEW parts!!
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8
OD
59
5
Time (hours)
Cd(II) 300 mM medium WT
Pconst.(H)-SmtA
Pconst.(L)-SmtA
Pconst.(L)-PduP1~18-fMT
BBa_K519022
BBa_K519023
BBa_K519018
BBa_K519014
E. coli with Pconst.(High) can grow well in high Cd2+ concentration than E. coli with Pconst.(Low).
Higher expression of SmtA and PduP1-18-fMT helps E. coli tolerate Cd2+ better. Metallothionein binds to
Cd2+ and prevents cytotoxicity. With metallothionein, E. coli can survive higher concentration of Cd2+.
Colonies of E. coli expressing phototaxis genes did not grow as big as controls,
indicating the phototaxis device successfully worked. Therefore we can control
E.coli movement by light.
