Contribution

Level 2 yeast shuttle vector

The yeast shuttle vector system, developed in partnership between the iGEM teams TU Dresden and WWU Münster, is an innovative vector class that will significantly simplify the work with Saccharomyces cerevisiae for all upcoming iGEM teams.

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 S. cerevisiae 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.

For the construction, the RFC1000 modular cloning system (MoClo) compatible vectors of the pSB1K0X and pSB3C0X series served as scaffolds. We, the iGEM Team Münster, focused on the development of the level 2 shuttle vector based on the pSB3C0X 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 S. cerevisiae 2µ ori was inserted.

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 aeBlue chromoprotein gene (Fig. 1) flanked by BsmBI class II restriction enzyme cutting sites. In the chromoprotein cassette, the aeBlue 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 aeBlue gene the vector was ready to use. Only the three class II restriction enzymes BsaI, SapI and BsmBI 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.

As the scaffold vector utilizes selection via the mRFP1-reporter module, our addition of the aeBlue now results in a deep purple staining of E. coli carrying the designed shuttle vector. Continuing from this, insertion of the selection marker leads to a red staining, while insertion of a MTU results in a blue staining of the cells. Finally, if both the selection marker and the MTU have been inserted, the colonies will not display any staining and hence appear white. This three-color selection scheme ensures easy identification of the plasmids state and hence eases handling and quality control.

Combining an exchangeable selection marker to keep the full capacity of the MoClo site, together with the easily recognizable three-color staining scheme, our system allows future iGEM teams to work with S. cerevisiae in an optimized manner, saving time, labor, and resources.

The aeBlue gene block

The aeBlue gene block represents a newly developed part of the yeast shuttle vector pSB3CY. The part was initially cloned in silico and synthesized via IDT. It consists of a SpeI and BsmBI recognition site and a BsmBI and NotI recognition site on the other. The CAP binding site and a ribosome binding site are flanking the promoter J23110 which have been additionally added for the translation of aeBlue, our protein of interest. The aeBlue is a chromoprotein that originates from the iGEM registry more precise from the Upsalla Chromoprotein collection. The translation is terminated by the double terminator rrnB T1 and the T7Te terminator. This constructed gene block is an improvement of the previous iGEM expression cassette containing the leaky promoter P_LacI and the standard red fluorescent protein (mRFP1), since the J23100 is not dependent on Isopropyl-β-D-thiogalactopyranosid (IPTG) to be active.

FSEOF-Software

The goal of metabolic engineering for industrial applications is the overproduction of metabolites with the help of microorganisms. While the field originated from single modifications in metabolic pathways, today’s approaches to metabolic engineering include a much more systematic view of biological systems. Computational tools based on flux balance analysis (FBA) of genome-scale metabolic models (GSMMs) have significantly advanced our capability to find non-obvious engineering targets for microbial cell factories. Most available software tools focus on predicting gene knockout effects and do not include the identification of putative gene amplification targets. Flux scanning based on enforced objective flux (FSEOF) is a prominent algorithm for identifying gene amplification targets and has been successfully used to optimize cell factories (Choi et al., 2010; Park et al., 2012). As no stand-alone FSEOF software tool is currently available and, to the best of our knowledge, no public code repositories can be found online, we decided to develop a user-friendly command line tool that utilizes the FSEOF algorithm for the identification of genetic overexpression and downregulation targets. The software is available at the iGEM GitLab repository collection. More information on the design considerations and the documentation of the software can be found on our software page.

BioReactor for Enzymatic ElectroSynthesis

Motivation

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.

Engineering of BREES

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.

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.

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.

We optimized our BREES to our needs but realized the potential of our design for several other applications (e. g. Biological Fuel-Cells, Biosensors) which for example need more air/gas exchange, multiple chambers, or different electrodes. Therefore, we designed different variations of the “receiver”, “coupling” and “cap” part resulting in a whole set of nine components which are compatible with each other. Future iGEM teams can thereby choose the right combination out of eight variations, depending on their experimental needs or expand the library themselves.

How to build BREES

High-pressure air can cause severe damage! 3D-printed parts can only handle low pressures. Only use the system with regulated low pressure!

1. Download the .stl files from the bottom of the page
2. Slice
• All parts do not need support structure
• Brims should only be used if bed adhesion is low
• Print temperature must be selected accordingly to the used filament
• We recommend slicing with Cura 5.0+
• Good quality can be achieved with a 0.3 mm nozzle and 0.12 mm layer height
• 40% infill
• Printing with TPU requires low printing speeds
• The receiver and cap can be printed together
3. Print (<6 h)
• Use a flat printer bed (e. g. spring steel-PEI)
• The printer bed needs to be leveled
4. Assembly
• Cleanup
• Insert hex nuts
• Insert electrodes
• Connect wire to electrodes with conductive glue
• Cure
• Screw together
5. Use
• Connect BREES to compressed air
• Fill with enzyme solution/ electrolyte
• Experiment…

CAD Files

click to download .stl