For our project, we aimed to design an open-source bioreactor suitable for the cultivation of E. coli and the
production of collagen-like proteins (CLPs).
Introduction
In the dynamic landscape of synthetic biology and biotechnology, bioreactors stand as essential tools, driving
innovation and advancing the frontiers of scientific exploration. These specialized vessels provide the
controlled
environment needed for the cultivation of microorganisms and the production of valuable bioproducts. For our
project, we aimed to design a bioreactor suitable for the cultivation of E. coli and the production of
collagen-like
proteins (CLPs).
Our initial designs
Our initial bioreactor design was quite similar to traditional continuously stirred tank reactors (CSTRs) and
to
designs of previous iGem projects (Leiden 2016 &
Exeter 2018). Although traditional
CSTRs
are commonly employed
in laboratory settings and their designs are well established, some of their limitations were revealed when
subjected to the demands of our project.
Fig. 1 An initial sketch of our bioreactor.
Fig. 2 An improved sketch of our bioreactor.
Redefining the bioreactor
Every modification made was a deliberate step towards creating a bioreactor better suited to our unique
requirements.
Our design choices, at a glance:
Oscillating Stirring Plate
+
Preserving product: Although E.coli can withstand shear stresses created from traditional impellers,
the
CLPs
are quite delicate, meaning their stability and functionality were at risk, so a gentler mixing method was
needed.
Achieving uniform mixing: In pursuit of more consistent and even mixing, we aimed to eliminate the
formation of
dead zones within the bioreactor, ensuring that every corner of the medium received equal attention.
Minimized foaming: Foam can take up valuable space in the bioreactor, reducing the effective working
volume for
the culture. Foam also reduces surface area available for oxygen exchange and it can result in excessive shear
stress within the culture medium.
Minimized vortexes: Vortexes can hinder effective mixing in the bioreactor as well as generate high
levels of shear stress and reduce oxygen transfer.
Electromagnet
+
This improves sterility as it eliminates the need for the multiple seals which come with the motor and sooner
or
later lead to contaminations. It also simplifies construction, reduces maintenance and increases room for
customization.
Removal of baffles
+
With reduced vortexes and foaming, the need for baffles was no more. This not only simplifies the construction
but also creates more space within the vessel, enhancing scalability and accommodating larger volumes.
Cooling and steam heating jackets
+
The original model only had a heater, but we introduced a cooling jacket and a steam heating jacket which
enables
us to fine-tune the growth conditions for our E.coli culture, ensuring optimal protein production. Although our
reaction is generally endothermic, introducing the cooling jacket ensures constant temperature throughout and
allows other uses for the bioreactor.
Our finished model
Fig. 3 Animation of our finished bioreactor model.
In this journey, we dive into the world of bioreactors, exploring their transformative potential for E. coli
cultivation and collagen-like protein production. With a reimagined design and a steadfast commitment to
precision,
we aim to unlock new possibilities in synthetic biology, one gentle stir at a time.
References
[1] Lehky, Pavel. "BIOREACTORS–NEW SOLUTIONS FOR OLD PROBLEMS." Lozibky, Brno, Czech Republic.
https://www.bioreactors.eu/files/bioreactor/minifor-bioreactors-article.pdf
[2] Glover, GM Cartland, and J. J. Fitzpatrick. "Modelling vortex formation in an unbaffled stirred tank
reactors." Chemical Engineering Journal 127.1-3 (2007): 11-22. https://doi.org/10.1016/j.cej.2006.09.019
We've created this protocol using the Protocol Designer Tool, utilizing various modules, pipettes, and
consumables for precision and accuracy:
P300 8-channel pipette
P300 single-channel pipette
Opentrons 4-in-1 tube rack with NEST 2 ml Screw Cap
NEST 12 well reservoir 15 ml
NEST 96 well plate 200 microliters flat
Opentrons 96 Tip Rack 300 microlitres
Opentrons 96 Tip Rack 20 microlitres
You can easily customize this protocol for different sets of pipettes and consumables by downloading the .json
file and importing it into the Protocol Designer Tool. Once customized, it can be seamlessly exported to the
Opentrons app for your specific research needs.
We present mathematical models to capture the gene expression archetype for collagen-like protein (CLP)
production
that would take place inside our open-source bioreactor.
Modeling the Rate of Production of Collagen-Like Protein (CLP)
We begin by considering the production rate of protein P with respect to time t:
\(\frac{dP}{dt}\) is the rate of change of P over time t.
\(\alpha\) is the production rate constant of P.
\(\lambda\) is the degradation constant for P.
The equation (Yildirim and Mackey 2003) accounts for both the protein production rate and its degradation. We
hypothesize that protein
production depends on mRNA concentration, with \(\alpha \cdot [mRNA]\) representing the production rate and
\(-\lambda \cdot P\) representing degradation. We acknowledge that protein degradation is natural, and we do
not
expect to extract 100% of the produced protein. Solving differential equation (1) for P and t, we obtain:
$$P = \frac{\alpha \cdot [mRNA] - e^{\lambda t }}{\lambda}$$
This equation assumes that at time \(t = 0\), the concentration of protein \(P = 0\). It can be used to
determine
the
concentration of protein P at any given time t. As the degradation rate of mRNA is faster than that of our
protein, we have included a linear equation for the concentration of mRNA to account for this:
$$[mRNA] = \beta - (\lambda + \delta) [mRNA]$$
Where:
\([mRNA]\) represents the concentration of mRNA.
\(\beta\) is the production rate constant for \([mRNA]\).
\((\lambda + \delta)\) is the decay rate constant for \([mRNA]\).
The term \((\lambda + \delta)\) highlights that mRNA degradation is faster than the degradation of
collagen-like
protein.
Modeling the Mixture of CLP and CLP-HyP
In our project, we employ Gene Code Expansion (GCE) to add Hydroxy Proline to Collagen-Like Protein (CLP).
This
crucial step in post-translational modification ensures the production of CLP with the correct folding. We
hypothesize that CLP-HyP, which contains an added hydroxy group, is prone to hydrolysis and dissociation into
CLP
with incorrect folding. The following equation describes this process:
$$\text{CLP-HyP} = \frac{P}{P + K_p}$$
Where:
\(P\) denotes the concentration of CLP.
\(K_p\) is the effective dissociation constant of CLP-HyP.
(Ilamaran et al. 2019) We assume that the effective dissociation constant of CLP-Hyp is small, and the majority
of produced CLP is
bound
to HyP and exhibits the correct folding.
Mathematical Models Describing the Lac Operon Model
In this section, we present equations modeling the Lac Operon. (Yildirim and Mackey 2003) This is relevant to
our project as we utilize
the
Lac operon model to express CLP. First, let's examine the binding of the Lac I repressor with the operator of
the
plasmid:
$$K(I) = \frac{K_0 \cdot I}{K_I + I}$$
Where:
\(K(I)\) is the effective dissociation constant of Lac I repressor-operator.
\(K_0\) is a constant.
\(K_I\) is the effective dissociation constant of Lac I repressor-IPTG.
\(I\) is the concentration of IPTG.
This equation illustrates the behavior of the Lac repressor in the presence of varying concentrations of IPTG.
It
can also be used to describe the production rate constant for protein. The production rate constant,
\(\alpha\),
is
not fixed but depends on the interplay between the Lac I repressor-operator interaction and the presence of
the
inducer molecule. The following equation represents this relationship:
$$\alpha = \frac{\alpha_0 K(I) K_0}{L + K(I)}
$$
Where:
\(\alpha\) is the production rate constant of \(P\).
\(\alpha_0\) is the initial production rate constant of \(P\).
\(K(I)\) is the effective dissociation constant of Lac I repressor-operator.
\(K_0\) is a constant.
\(L\) represents the concentration of Lac I repressor.
As the effective dissociation constant \(K(I)\) approaches infinity (indicating weak binding of the Lac I
repressor
to the operator), \(\alpha\) reaches its maximum value. In practical terms, this suggests that when the Lac I
repressor barely binds to the operator, transcription proceeds uninhibited, leading to the highest possible
production rate of CLP.
References
Yildirim, Necmettin, and Michael C. Mackey. “Feedback regulation in the lactose operon: A mathematical
modeling study and comparison with experimental data.” Biophysical Journal, vol. 84, no. 5, 2003, pp. 2841–2851,
https://doi.org/10.1016/s0006-3495(03)70013-7.
Ilamaran, Meganathan, et al. “A Self-Assembly and Higher Order Structure Forming Triple Helical Protein as a
Novel Biomaterial for Cell Proliferation.” Biomaterials Science, vol. 7, no. 5, 23 Apr. 2019, pp. 2191–2199,
pubs.rsc.org/en/content/articlelanding/2019/bm/c9bm00186g#, https://doi.org/10.1039/C9BM00186G. Accessed 4 Nov.
2020
In a Nutshell
A Synthetic Biologist’s Guide to Novel Foods is the culmination of our research on the topic of novel
foods,
with contributions from our Human Practices interviewees — experts in food safety and fermentation. In addition
to it being a collection of our research, the Guide also serves as a resource for the current and future iGEM
teams across many tracks — from Biomanufacturing to Food and Nutrition — who choose to focus on ingestibles.
A major obstacle for us was importing the chemicals to the United Arab Emirates, as such we have made the UAE
Chemical Importing Guide outlines the steps required to import chemicals and minimize time delays. As the only
iGEM team in the UAE we have written this guide to be a resource for other iGEM teams and researchers in the
UAE. In some cases, the shipping and handling fee along with the customs duty can greatly increase cost. For
instance, the E. Coli auxotroph strains were USD 103 each, handling charges were USD 200, freight charges were
USD 1100, Ministry of Health permit was USD 477.83, and duty charges were USD 150. We were fortunate to have the
NYUAD logistics team assist us regarding the import procedure. We’ve included the format to the End User
Declaration and MOHP Permit letter as part of our contribution. Another suggestion to future teams would be to
order oligos and other nucleic acid as lab ready formulation rather than dried form when ordering from IDT.
Dried nucleic acids took much longer and needed separate permits.
