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Cellfiefuge
An
auto-pelleting system
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height="60" width="320" />
<a
href="#description">Description</a>
</svg>
<a
href="#overview">Overview</a>
<a
href="#design">Design</a>
<a href="#protein">Protein
production module</a>
<a href="#aggre">Auto Aggregation
module</a>
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Since the advent of rDNA
technology in the late 1970s, the
use of recombinant protein has been
on the rise, starting from insulin
and hormones all the way through to
monoclonal antibodies and one
hundred and fifty-one FDA approved
protein based recombinant
pharmaceuticals by the year 2009[1].
Despite being in high demand,
recombinant proteins have heavy
price tags, thanks to the
significant manufacturing costs. One
of the important steps in the
manufacturing process is
Centrifugation but on an industrial
scale, it is an economic nightmare.
[2] Further, existing industrial
setups use complex sensors to
monitor cell density to determine
the point at which to begin
separation of the cells from the
cell culture. One can immediately
see the humongous relief that would
be associated with finding a way to
deal with the above problems. Hence,
we came up with the idea of our
project, the Cellfifuge.
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We realized that
manufacturing units use expensive
and energy expensive machinery [3].
Hence, we engineered our bacteria in
such a way that it could do most of
the work done by these monstrous
machines so that we no longer have
to buy them and pay their bills.
To
increase the yield of the product,
we came up with the idea of
separating cell growth and target
protein production so that when the
cells have grown up to a high enough
cell density, they shuttle all their
energy towards the required protein
production.[4] Based on the above
ideas, we came up with the design of
our bioreactor. (Figure 1)
We realized that we would not be
able to work on the whole idea this
summer so, we focussed on two
modules of our design: the Protein
production module and the
auto-aggregation module leaving the
arresting of cell growth for future
iGEM teams as a seed for their
ideas.
<img
class="image" src="designa.jpg"
/>Figure 1: The bioreactor design of
Cellfifuge
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Induction synthesis of protein
of interest is another important
step. Microorganisms in bioreactors
are induced at the OD, at which the
protein production is maximum. [5]
Because of this, factories install
sophisticated machinery to keep
track of the OD value of the culture
so as to know when to induce the
production. We wanted our bacteria
to know when to start protein
production and start it by itself.
Hence, we used the lux quorum
sensing system to sense this cell
density. [6] We have expressed the
gene for our protein of interest
under the luxI gene so that protein
of interest is produced in
significant quantities only when the
quorum is achieved.(Figure 2) We
also wish to tune the quorum sensing
system by tweaking with the strength
of the promoters involved in quorum
sensing.
<img class="image" src="lux.jpg"
/>Figure 2 : The lux quorum sensing
system. Note the gene of interest
"geneX*" downstream of the luxI
gene. Image Courtesy: ETH Zurich
2013, iGEM team.
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To achieve cellular
aggregation, we use the famous
auto-transporter protein, Antigen43
(Ag43). [7] It has shown to cause
auto-aggregation of cells when
overexpressed. (Figure 3) We plan to
exploit this property of Ag43 to
substitute centrifugation. We
acknowledge previous iGEM teams who
have worked on Ag43 (Hokkaido
University, 2012; Aberdeen Scotland,
2014 and some more) as we got the
BioBricks for Ag43 readily
available. We intend to express
Ag43 only when the cells have made
the product of interest. Hence, we
use the diauxic shift in the media
to act as a switch for the
expression of Ag43.
<img class="image"
src="tube.jpg" />Figure 3: a) Cell
culture without expression of Ag43
b) Cell culture overexpressing
Ag43
Source: MarjanW. van derWoude
et al, 2008
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- Ferrer-Miralles N,
Domingo-Espin J, Corchero JL,
Vazquez E, Villaverde A. Microbial
factories for recombinant
pharmaceuticals, Microbial Cell
Factories, 2009:8:17.
- Medipally S.R., Yusoff F. Md.,
Banerjee S., Shariff M. Microalgae
as sustainable renewable energy
Feedstock for Biofuel Production,
BioMed Research International, 2015.
- Felo M.,
Christensen B., Higgins J., Process
cost and facility considerations in
the selection of primary cell
culture clarification technology,
AIChE, 2013.
-
Motoo Suzuki, Rohini Roy, Haiyan
Zheng, Nancy Woychik, Masayori
Inouye, Bacterial Bioreactors for
High Yield Production of Recombinant
Protein. The Journal of Biological
Chemistry, 2006.
-
Rosano G.L., Ceccarelli E. A.,
Recombinant protein expression in
Escherichia coli: advances and
challenges, Frontiers in
Microbiology, 2014.
- Miller M.B., Bassler B.L.,
Quorum Sensing in Bacteria, Annual
Review of Microbiology, 2001.
- Marjan W. van der
Woude, Ian R. Henderson, Regulation
and Function of Ag43 (Flu), Annu.
Rev. Microbiol., 2008.
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