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Commit f0ca5a84 authored by Marius Luttermann's avatar Marius Luttermann
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<p>The idea to engineer this vector system arose from a necessity. Our two teams realized, that the vectors iGEM had provided were not suitable to perform all the experiments the way we had envisioned. Thus, we gathered together in several meetings, where we were able to elaborate our idea of a <i>S. cerevisiae</i> shuttle vector system. Easy handling and flexibility were the most important goals for us. To achieve our aim, incorporating an appropriate origin of replication as well as an effortlessly replacable yeast-specific selection marker were prerequisites. </p>
<p>For the construction, the RFC1000 modular cloning system (MoClo) compatible vectors of the <i>pSB1K0X</i> and <i>pSB3C0X</i> series served as scaffolds. We, the iGEM Team Münster, focused on the development of the level 2 shuttle vector based on the <i>pSB3C0X</i> series. To start with the engineering, the vector was linearized using Gibson primers for a specific site. This site was chosen to be located opposing the MoClo cloning site harboring the red fluorescent protein (RFP)(Fig. 1) reporter module. After linearization, the well-described <i>S. cerevisiae</i> 2µ ori was inserted. </p>
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<img style="width: 100%" src="https://static.igem.wiki/teams/4188/wiki/wiki-images/shuttlevektoren-contribution.png" target="_blank">
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<figcaption><b>Figure 1: Colonies colored by mRFP1 and aeBlue.</b> <i>Escherichia coli</i> colonies with our shuttle vectors.</figcaption>
</figure>
<p>As flexibility was one of our main goals, we introduced a new site for an exchangeable, yeast specific selection marker as the heart of our shuttle vector system. In our design, a lot of small fragments were combined to form a new cloning site, harbouring the <i>aeBlue</i> chromoprotein gene (Fig. 1) flanked by <i>Bsm</i>BI class II restriction enzyme cutting sites. In the chromoprotein cassette, the <i>aeBlue</i> gene was put under a constitutive promoter and a double terminator. This designed construct was synthesized for us by Integrated DNA Technologies (IDT). After insertion of the <i>aeBlue</i> gene the vector was ready to use. Only the three class II restriction enzymes <i>Bsa</i>I, <i>Sap</i>I and <i>Bsm</i>BI are necessary to successfully equip the vector with the desired parts (Fig. 2). This design allows for specific exchange of the selection marker via Golden Gate Assembly, and is one of the unique and great advantages our system has to offer. In addition, the full capacity of the multi-transcription unit (MTU) MoClo site could thus be preserved. This increases flexibility, as assembly of MTUs can be time-consuming and set MTUs cannot be changed in order to incorporate a selection marker afterwards. </p>
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<figcaption>
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<b>Figure 2: Map of pSB3KY-aeBlue. </b>
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<h4>Motivation</h4>
<p>We developed a 3D-printed BioReactor for Enzymatic ElectroSynthesis (BREES). Our approach to Enzymatic ElectroSynthesis (EES) requires the immobilization of enzymes on an electrode. A common way of doing this is by using a peptide linker with an affinity to negatively-charged surfaces like glass or indium tin oxide (ITO) (Zernia et al., 2018). In recent studies, ITO-electrodes for EES were custom made by a lift-of-technique (sputtering) on glass plates (Frank et al., 2020). This approach requires very expensive production capabilities and know-how, so we had to settle on commercially available ITO-electrodes. These electrodes have no busbar, can easily be destroyed by scratches, and are only coated with ITO on one side. To overcome these problems, we developed a 3D-printed bioreactor to hold and contact the ITO-electrode, as well as a counter-electrode (platinum) and a reference-electrode. Within several engineering circles we established a system which can be connected to pressurized air to prevent leaks, ventilate, and stir the electrolyte. It can be driven with an Arduino-based potentiostat (Crespo et al., 2021) to build an inexpensive platform for all kind of electrochemical or enzymatic electrosynthesis projects.</p>
<h4>Engineering of BREES</h4>
<a href="https://static.igem.wiki/teams/4188/wiki/wiki-images/brees-development-min.png" target="_blank">
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<figcaption><b>Figure 3: Different generations of BREES.</b> After testing multiple very different designs, we settled on the current modular system and started improving it.</figcaption>
</figure>
<p>We designed BREES around a commercial ITO-electrode. The first design challenge for this set-up was to design a system including the electrodes and wire to enable the attaching of the ITO-electrode onto an electric circuit. </p>
<p>For this, we decided to design a bioreactor which is simple to build, inexpensive in production, and easy to adjust (Fig. 3). To face these challenges, we used 3D-printing technology, specifically the Fused Filament Fabrication (FFF). BREES is composed of three different 3D-printed parts, which allows a modular set-up with different materials. The bottom part (“receiver”) is printed from polylactic acid (PLA) to hold the ITO-electrode and contain the hex nuts used for screwing the parts together. PLA is very easy to print and the standard choice for 3D-printing rigid structures. The top part (“cap”) is also made of PLA. It holds a counter-electrode and connects BREES to a compressed air hose. Some versions can hold a reference-electrode. Furthermore, it can be screwed on the lower parts to fix them in place. The middle part (“coupling”) is printed from thermoplastic polyurethan (TPU), which is rubber-like but more difficult to print than PLA. Thereby, it can be used as a tight seal between the receiver and cap part and prevents leakage of the electrolyte. This middle part defines the distance between the electrodes and the electrolyte volume. It also separates a corner of the lower electrode from the electrolyte to allow electric contacting of the electrode via a water based electrically conductive paint and a copper wire.</p>
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<img style="width: 100%" src="https://static.igem.wiki/teams/4188/wiki/wiki-images/brees-compressed-air.png" target="_blank">
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<figcaption><b>Figure 4: BREES in action.</b> The Bioreactor is sealed, stirred and ventilated by compressed air as indicated by the bubbles.</figcaption>
</figure>
<p>All parts of BREES have small, compressed air tubes included, so a positive pressure differential around all edges prevents leakages without additional components (Fig. 4). This particular property is the result of several engineering cycles, in which we tried several options to prevent leakage of our electrolyte. By using air pressure, we can prevent contamination and substrate absorption which were the result of using silicon oil. This system also allows usage of a variety of solvents if electrodes and printing materials are selected accordingly. The modular system can be easily reassembled after cleaning or exchanging of single parts. This makes the system suitable for versatile applications and optimization of the surface to volume ratio by changing the size of the electrolyte containment. It also allows the usage of different electrodes by simply changing the cap. The parts are either for the horizontal or vertical set-up and must be chosen accordingly.</p>
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<br>
<h3>Design</h3>
<p>We engineered the yeast’s mevalonate pathway to overcome its rate-limiting steps. By introducing additional
heterologous genes and silencing endogenous genes, we modified the pathway to suit our needs. [<a class="link-dark"
href="https://2022.igem.wiki/wwu-muenster/results#sccytosol">Cytosolic production of α-pinene in Saccharomyces
cerevisiae</a>] Our goal was to increase geranyl diphosphate (GPP) production in <i>Saccharomyces cerevisiae</i>, as it acts as an essential precursor for monoterpenoid synthesis such as α-pinene. Hence, we
heterologous genes and silencing endogenous genes, we <a class="link-dark"
href="https://2022.igem.wiki/wwu-muenster/results#sccytosol">modified the pathway to suit our needs</a>. Our goal was to increase geranyl diphosphate (GPP) production in <i>Saccharomyces cerevisiae</i>, as it acts as an essential precursor for monoterpenoid synthesis such as α-pinene. Hence, we
engineered the cytosolic pathway, but also targeted another cell compartment. Since physiological conditions in
peroxisomes are favorable for GPP production, we hypothesize that an increased α-pinene concentration is observed if
the corresponding synthesis pathway is relocated there.</p>
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<h4>Materials and Methods</h4>
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For our shared design, our teams decided for a prior insertion of the 2µ origin of replication (<i>2µ ori</i>), ensuring the high-copy plasmid replication inside of <i>S. cerevisiae</i>.
Allowing easy, efficient, and variable insertion of auxotrophy markers, we used a Golden Gate Assembly system with a blue chromoprotein (<i>aeBlue</i>) mediated selection process.
The new chromoprotein cassette can be replaced via Golden Gate Assembly with the restriction enzyme <i>Bsm</i>BI and exchanged for the desired selection marker. The combination of two chromoproteins can be seen during cultivation by a color reaction that visualizes the genetic insertions into the corresponding sites. While the initial vector shows a purple color, successful transformants turn red if an auxotrophy marker is added via Golden Gate Assembly. Furthermore, the insertion of a successful Golden Gate Assembly with an MTU into the site of the red fluorophore (<i>mRFP1</i>) gene the vector shows blue staining of the colonies. If both chromoproteins are exchanged, the transformants become white and can be transformed into <i>S. cerevisiae</i> after plasmid extraction.
If a change of the plasmid is required after successful Golden Gate Assembly, the selection marker can be exchanged retrospectively using the restriction enzymes <i>Not</i>I, <i>Spe</i>I, and <i>Bam</i>HI (Fig. 1). For further information please look at our <a class="link-dark" href="https://2022.igem.wiki/wwu-muenster/contribution">contributions page</a>.
If a change of the plasmid is required after successful Golden Gate Assembly, the selection marker can be exchanged retrospectively using the restriction enzymes <i>Not</i>I, <i>Spe</i>I, and <i>Bam</i>HI (Fig. 1). For further information please look at our <a class="link-dark" href="https://2022.igem.wiki/wwu-muenster/contribution#section-1">contributions page</a>.
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<p>
The iGEM Team TU Dresden received our <i>pSB3CY-aeBlue</i> plasmid, to introduce <i>aeBlue</i> into their level 1 shuttle vector. While they provided us with their level 1 <i>pSB1KY</i> plasmids, which we used for the expression of several genes. The introduction of the <i>pSB1KY-URA3-ApL3H</i> into an α-pinene producing strain led to the conversion from α-pinene to verbenone, our desired final product.
Additionally, we cloned the <i>afraGFP</i> gene with and without the C-terminal SKL peroxisomal targeting signal-1 (PTS1) into the <i>pSB1KY-HIS3</i>, through which we could successfully characterize the PTS1 tag and confirm the localization of proteins into the peroxisome instead of the cytosol (Fig. 2) For further information please look at our <a class="link-dark" href="https://2022.igem.wiki/wwu-muenster/engineering">engineering success page</a>.
Additionally, we cloned the <i>afraGFP</i> gene with and without the C-terminal SKL peroxisomal targeting signal-1 (PTS1) into the <i>pSB1KY-HIS3</i>, through which we could successfully characterize the PTS1 tag and confirm the localization of proteins into the peroxisome instead of the cytosol (Fig. 2) For further information please look at our <a class="link-dark" href="https://2022.igem.wiki/wwu-muenster/engineering#section-2">engineering success page</a>.
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<a href="https://static.igem.wiki/teams/4188/wiki/wiki-images/partnership-4-fig-2.png" target="_blank">
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