Partnership

Shared design of yeast shuttle vectors

We aimed at a shared design for our shuttle vectors to create an easy MoClo-based system (RFC1000 assembly standard) for future iGEM teams. This system is not only highly versatile, but also allows for simplified assembly and facilitates selection of positive transformants through two different reporter proteins. Additionally, the destined site of insertion for a yeast selection marker gene, is compatible with iGEMs Open Yeast Collection, so that future teams can choose from a wide variety of genes. The shuttle vectors were development based on iGEM standard vectors pSB1K04 (level 1) and pSB3C01 (level 2). The new shuttle vectors allowed us to express genes in Saccharomyces cerevisiae, which was not possible on level 1 plasmids prior to our work. To express genes on level 2, one position of the multi-transcription unit (MTU), containing four transcription units in total, was required to be a selection marker for S. cerevisiae preventing the introduction of four genes on one plasmid. Since we integrated several genes for the heterologous mevalonate pathway in peroxisomes on a level 2 shuttle vector, we created a vector that contained a selection marker independent of the MTU, to increase the vectors capacity. For our shared design, our teams decided for a prior insertion of the 2µ origin of replication (2µ ori), ensuring the high-copy plasmid replication inside of S. cerevisiae. Allowing easy, efficient, and variable insertion of auxotrophy markers, we used a Golden Gate Assembly system with a blue chromoprotein (aeBlue) mediated selection process. The new chromoprotein cassette can be replaced via Golden Gate Assembly with the restriction enzyme BsmBI 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 (mRFP1) gene the vector shows blue staining of the colonies. If both chromoproteins are exchanged, the transformants become white and can be transformed into S. cerevisiae 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 NotI, SpeI, and BamHI (Fig. 1). For further information please look at our contributions page.

The iGEM Team TU Dresden received our pSB3CY-aeBlue plasmid, to introduce aeBlue into their level 1 shuttle vector. While they provided us with their level 1 pSB1KY plasmids, which we used for the expression of several genes. The introduction of the pSB1KY-URA3-ApL3H into an α-pinene producing strain led to the conversion from α-pinene to verbenone, our desired final product. Additionally, we cloned the afraGFP gene with and without the C-terminal SKL peroxisomal targeting signal-1 (PTS1) into the pSB1KY-HIS3, 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 engineering success page.

In summary, we created a collection of versatile and efficient shuttle vectors together in our partnership with the iGEM Team TU Dresden, useful for all future iGEM teams working with the popular model organism S. cerevisiae.

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Thymol production and application

Throughout our partnership with the iGEM Team TU Dresden, we found another connection between our projects: MonChassis provides a manufacturing platform for WunderBand. The iGEM Team TU Dresden developed a hydrogel system containing bacteriophages, applicable for the treatment of chronic wounds. In search of adjuvants for their hydrogel, specifically anti-inflammatory agents, we found many monoterpenoids to be already used as pharmaceuticals. We figured that the monoterpenoid thymol would be best suited to be embedded into their hydrogel. Thymol was shown to significantly reduce oedema in rodents and reduced the influx of leukocytes into the wounded area. When wounds were treated with a collagen-based thymol saturated film, the wound retraction rate was significantly higher, the granulation reaction was improved and the collagenization density and arrangement during wound healing was improved (Riella et al. 2012). Thymol released from nanostructured lipid carriers could improve wound healing and inflammation of the inflammatory skin disease psoriasis induced in mice (Pivetta et al. 2018). Additionally, thymol decreased the production of elastase, a marker for inflammatory diseases, in human cells, which protects the wound tissue (Braga et al. 2006). The anti-bacterial properties of thymol have been described several times. Xu et al. (2018) showed the elimination of colony forming units of Escherichia coli within 6 hours by adding 400 mg/l thymol to liquid MH media. The well documented anti-inflammatory and anti-bacterial properties made thymol a good candidate, also due to its hydroxy group and the resulting higher polarity, compared to other molecules, it would likely be able to be incorporated into the hydrogel platform of the iGEM Team TU Dresden, which they could confirm experimentally. In Thymus vulgaris, GPP is converted to γ-terpinene by the γ-terpinene synthase 1 (TvGTPS1), it is oxidized by a cytochrom P450 monooxygenase (CYP) of the 71D family (TvCYP71D179) to a dienol intermediate. This intermediate is reduced by the short chain dehydrogenase 1 (TvSDR1) to an instable allylic ketone intermediate which rearranges to thymol (Fig. 3) (Krause et al. 2021).

Due to the versatility of MonChassis, we could simply replace the last synthesis steps from GPP or neryl pyrophosphate (NPP), and introduce the three proteins needed for thymol synthesis. We decided to introduce these three genes into our respective yeast strains that were optimized for NPP/GPP production. The NPP producing strains being especially interesting, since it is not known if the TvGTPS1 can utilize NPP as educt. We aimed to introduce the three genes into either the level 2 plasmid pSB3C01 or into a level 2 shuttle vector, and cloning for the plasmids was carried out by all four yeast subgroups. Due to challenges in generation of GPP/NPP-optimized strains and time constraints, just the peroxisomal approach in S. cerevisiae could have produced results. Unfortunately, transformations with the shuttle vector containing the genes necessary for thymol synthesis did not yield any colonies. Even though we were not able to complete transformation the general working principle was shown by Krause et al. (2021), reporting thymol production in in S. cerevisiae. Our approach is therefore likely to produce thymol, possibly in higher quantities than described before. To test the medical application of thymol, the iGEM Team TU Dresden successfully incorporated 2.5 mg/ml, 5 mg/ml, and 10 mg/ml thymol into their hydrogel, and carried out some tests on the efficacy of the release of thymol from the hydrogel and simultaneously its anti-bacterial function on plated E. coli. As depicted in Figure 4 A no E. coli colonies could grow in a circle around the hydrogel soaked in 5mg/ml and 10 mg/ml thymol, suggesting that thymol was released, and it inhibited the growth of E. coli. The released thymol showed a greater anti-bacterial effect than ethanol/phosphate-buffered saline (PBS) (1:1 v/v) (Fig. 4B). For more information about the hydrogel platform and the tests made, please visit the partnership page of the iGEM Team TU Dresden.

The partnership with the iGEM Team TU Dresden supported us in the execution of essential project details, as the pSB1KY shuttle vector, cloned by them with our shared design, was crucial for verbenone production within S. cerevisiae. The stimulating and incisive discussions led to overall improved lab work. We were not only able to benefit from each other, but we created a collection of S. cerevisiae shuttle plasmids for variable and eased gene insertion screening, accessible to all future iGEM teams. Even though we did not successfully finish transformation of thymol producing enzymes, initial trails pointed out that MonChassis can indeed be used as a versatile and adaptive modular platform.