PosterPDF Available

Abstract

Nanoparticles can be used for a wide range of applications. Especially if used in the medical field they need to be free from toxic chemicals, which are used in the conventional way of nanoparticle formation. Therefore, our goal is to use synthetic biology to create a way of generating environmentally friendly nanoparticles. Our Escherichia coli take up metal ions like iron, copper, silver or gold ions to accumulate them in the periplasm and the cytoplasm. To prevent metal ion export, several genes need to be silenced. Besides using knock-out strains, which are created using CRISPR/Cas9, we aim to introduce a two-plasmid system working with siRNA to ensure a essential minimal expression of some genes. In addition, our E. coli they have several optimized mechanisms against the toxicity of metal ions. Within the cell the ions get accumulated and reduced to nanoparticles. These nanoparticles will be isolated from the cells and could be used in different way. Our proof of concept will be the printing of electric curcuits for electronic tattoos out of nanoparticle ink consisting mainly of our self-produced nanoparticles.
Team Members (from left
to right) Erika Schneider,
Matthias Otto,
Antonin Lenzen,
Jakok Zubek,
Christoph Geske,
Levin Joe Klages,
Johannes Ruhnau,
Vanessa Krämer,
Irina Rais
[1] Scott, R. L., & Hays, R. M. (1975). Inactive and abandoned underground mines: water pollution prevention and
control (Vol. 75, No. 7). US Environmental Protection Agency.
[2] Zhao, G., & Stevens, S. E. (1998). Multiple parameters for the comprehensive evaluation of the susceptibility of
Escherichia coli to the silver ion. Biometals, 11(1), 27-32.
[3] Li, F. et al (2013). Analysis of copper nanoparticles toxicity based on a stress-responsive bacterial biosensor
array. doi: 10.1039/c2nr32156d
References
With great thanks to our many advisors and our PIs:
Julian Droste, Carsten Hain, Marten Linder,
Janina Lüders, Boas Pucker, Pascal Schmidt,
Svenja Vinke
Acknowledgements
Supporters
Dr. Christian Rückert and Prof. Dr. Jörn Kalinowski
nano
Introduction
Several strains of Pseudomonas spp. are able to import heavy metal ions. OprC is a
putative transporter for copper ions into the periplasm. Optional transport into the
cytoplasm can be achieved by HmtA. Exporters will be silenced by our two plasmid
siRNA system.
Known reductases like MerA and NiR can be
used in order to produce mercury and silver
nanoparticles. By using mutagenesis MerA can
be modified so that it recognizes other metal
ions such as copper ions.
Ferritin is a storage protein for iron
ions. It forms iron oxide
nanoparticles and helps to
accumulate iron in the cell.
Ferritin
As a proof of concept, we print electronic circuits based on nanoparticles. As another
approach nanoparticles can be loaded with siRNAs for further application.
Applications
Figure 8: A desktop inkjet printer, which
can be used to print electronic tattoos.
Reductase
oxidative
stress
Cu(I,II), H2O2
Cu(I,II), H2O2
CuNPs
damageDNA
protein
damage
damage
membrane
ROS
ROS
Toxicity
Nanoparticle
Nanoparticles are accumulations of atoms with a size of a few nanometers. Production in cells makes them clinically
relevant due to the absense of toxic solvents which are used in most chemical synthesising methods.
ERROR 404
80 - 200 °C
pTed promotor
AmilCP
(reportergen)
LacZ_Alpha_BbsI
Linker
pSi_Reporter
araC
Hfq scaffold
araBAD promotor
LacZ_Alpha_GGA
pSi_Target
Ferritin
Fe2+
Fe2+
Fe2+
Fe2+
Fe2+
Fe2+
target
mRNA
siRNA
RNase
Hfq
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siRNA
RNase
Hfq
target
mRNA
siRNA
Hfq
target
mRNA
siRNA RNase
Hfq
target
mRNA
genomic
DNA Gene1 Gene3
genomic
DNA Gene1 Gene3Gene2
genomic
DNA
Cas9
Gene1 Gene3Gene2
Cas9
Toxicity-Curves
Printing
Conductive patterns can be printed with inks consisting of
24 wt% of NPs and 76 wt% of organic solvent. Attached to the
skin they can measure personal vital signs like heartbeats or
even brainwaves.
Functionalized Magnetic NP
Fe2O3 NP are ferromagnetic and when functionalised new
applications like the penetration of biofilms become possible.
By attaching our own si-RNAs to
such an magnetic particles we hope
to make them even more useful for
clinical applications.
The generation of Cu(I)-ions [3] and hydrogen peroxide by Cu-
nanoparticles (NPs) results in damage to proteins, DNA and membranes
(Figure 2).
As intracellular metal ions benet the formation of reactive oxygen
species (ROS), overexpression of antitoxic agents like phytochelatin,
which is rich in sulfhydryl groups, are used to tackle this problem.
The eects of Cu-NPs on
the growth of E. coli was
much less severe than
suggested by the literature
probably due to NP
agglomeration (Fig. 4).
Silver ions are very toxic to
E. coli while copper ions can
be handled better by the
cell (Fig. 3 and 5).
Figure 1: Copper (Cu) transport systems in E. coli: Cu(I)-ions are exported out of
the cytoplasm by the CopA-ATPase. Cu(I)-ions are exported from the
periplasmatic room to the outside of the cell by the antiporter CusCBA [1].
OprC is a TonB-dependent receptor that transports specificly copper ions and
assumingly reduces Cu(II)-ions simultaneously [2]. To transport Cu(II)-ions into
the cytoplasm, the P-type ATPase HmtA is used.
Figure 3: Growth with different Ag(I)-ion concentrations.
Figure 4: Growth with different CuNP concentrations. Figure 5: Growth with different
Cu(II) ion concentrations.
We organized a talk regarding the topic of Dual
Use Research of Concern. For that purpose,
we collaborated with the btS e.V. GS Bielefeld
and invited experts. Furthermore, we created
a survey on the same topic to find out how well
informed scientist are regarding to that topic.
putative
transporter
CopA
Cu(I)
CusCBA
Cu(II)
Cu(I)
Cu(II)
Cu(I)
H+
ATP
ADP + P
H+
CusC
OprC
Cu(II)
Cu(I)
TonB
HmtA
Cu(I)
PA5030
Cu(II)
Cu(I)
Cu(II)
Figure 2: Interaction of Cu(I,II)-ions with ROS and the resulting cell damages.
Figure 6: Two-plasmid system for expression and testing of siRNAs in compasison with CRISPR/Cas9
Figure 7: Storage of Fe(III) as Fe(II)
in Ferritin and formation to NPs
Taking up metal ions and reducing them inside
Escherichia coli is the core of our project. Afterwards,
elemental metal agglomerates to nanoparticles which
can be used in various ways due to their special
properties.
At the moment our focus lies on the uptake of copper
and iron ions. The knockout of various copper exporters
with CRISPR/Cas9 is lethal to the cells so we decided to
work on a siRNA-Toolbox to silence the synthesis of
unfavourable proteins.
Importer/Exporter
Knocking out genes can significantly lower
the growth rate. As an alternative genes
can be silenced by using siRNAs or RNA-
interference (RNAi).
Establishing a two-plasmid system for
expression and testing of siRNAs and
siRNA libraries can be used to compare
the silencing ability of several siRNAs by
measuring fluorescence.
si-RNA
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