Revision as of 00:48, 13 December 2019

Parts

During our journey of making our yeasts fantastic, we built many blocks which we wish to make reusable to further iGEM teams. We have packaged our main system for directed evolution, OrthoRep, with all the means to evolve your own proteins of interest. Furthermore, we provide a set of parts for engineering modular, orthogonal GPCRs in yeast. All in all, we provide a toolbox of parts containing:

The error-prone DNA polymerase from K. lactis used for OrthoRep (TP-DNAP1).
An extensible integration cassette for evolving your own parts using our system.
A modular toolbox for building your own orthogonal GPCRs in S. cerevisiae

Best basic: polymerase BBa_K3173000

This part (terminal protein DNAP1) represents a DNA polymerase which was extracted from the K. lactis – a yeast strain actively used in biotechnology (especially fermentation) and science [Bergquist2002]. This strain is also known to harbor and express a linear cytoplasmic yeast plasmids.

This part is an element of the Orthorep system, where it is responsible for replicating the engineered plasmid with the genes of interest. We used a yeast strain harbouring the aforementioned polymerase and linear plasmid, which was developed for increased error rate of the polymerase and characterized by the group of Chang Liu Zhong2018. Since the common yeast strains employed in research (S. cerevisiae) are lacking linear cytoplasmic plasmids in normal conditions, the insertion of this polymerase, together with the engineered OrthoRep system allowed us to orthogonally mutagenize genes of interest in vivo [Ravikumar2018].

To better understand the system, we first sought to take a look at the structure of the polymerase. As for general characteristics, the polymerase consists of 995 amino acids and has a mass of around 116 kDa. A search for more structure-related information in the literature turned up blank, and even finding our protein in Uniprot left us without a structure. Thus, we had to take matters into our own hands and attempt to solve the structure of TP-DNAP1. We proceeded to build homology models of the protein using SwissModel.

There, we were met with a pitiful amount of homology to our protein, which reliably drove the model quality into the ground. Another difficulty arose due to the lack of homological structures for a large part of the C-terminus of our protein (first 325 AA were thus not included in the model, that correspond to the terminal protein sequence). Now, we had two options left: solve our structure de novo or crystallize it, and we sure as heck wouldn't go for the last option. Therefore, we moved on to greener pastures and submitted our sequence to both Robetta and RaptorX. The results of the de-novo structure assembly can be seen in the images below. According to the obtained images our polymerase has 4 domains including the capping protein. Here we present only the actual polymerase domain.

Best composite: BBa_K3173001

Here we present to our best composite part – the OrthoRep integration cassette. This part is the key to speeding up the evolution of your gene of interest. As the name already implies, this cassette integrates into the OrthoRep system, where all the genes on it get rapidly mutagenized by the engineered error-prone fungal orthogonal DNA polymerase. The cassette as presented here consists of several parts, for example the sequences needed for integration into the cytoplasmic plasmid which allows the system to be fully orthogonal to the rest of the yeast genome. Further, it contains several selection markers.: For instance the ampicillin resistance gene, allowing for easy selection of the modified cassette in bacteria, as well as leu2 and ura3 genes, which make it possible to first, maintain the plasmid in yeast and secondly to select for transformed colonies via standardized auxotrophy screenings (selection on drop-out media).

Liza, [22.10.19 05:50]

Before proceeding with integrating our gene of interest we first characterized all the reported functions of the integration cassette. Therefore, the cassette was transformed into E.coli and grown on an ampicillin LB agar plate. Then we moved to portray the plasmid behavior in yeast. For that, the F102 strain containing 3 auxotrophies (-leu2, -his2, and -ura3) was supplemented with the OrthoRep cassette and grown in different conditions. After characterizing the leu2 gene dependency we continued to study the most tunable selection marker of the cassette – the ura3 gene. Thereby we first went for describing the yeast growth in various concentrations of uracil. The results can be seen in the following graph.

Next, we moved to generate a library of ura3 mutants using the selective pressure of 5-FOA. Here, the colonies were kept in the delta Leu medium for minimizing the selective pressure on ura3, thus allowing them to mutate the gene. After several weeks the colonies were transferred onto 5-FOA containing media to see if growth still occureds, which would indicate the successful mutation of the gene. The results of the experiment can be seen in figure.

As one can see, the complete inactivation of ura3 even without selective pressure took multiple weeks to obtain. But once achieved it remained relatively stable. Possible effects contributing to this result might be first the inhibition of nucleotide synthesis by 5-FOA, which "conserves mutations", as well as the large threshold due to multiple plasmids being present in the cell at the same time. The more complete explanation with the corresponding calculations can be obtained from our model. The observed effects prove ura3 to be an optimal selection marker for more complex evolving systems, which need an internal, easy to measure control for the mutation rate (which can be calculated using our model).

characterization: polymerase BBa_K3173004

Leu2 is a gene coding for the enzyme 3-isopropylmalate dehydrogenase (also known as leu3) that on its turn is involved in the synthesis pathway of L-leucine. The gene presented here originates in yeast (S. cerevisiae), however, its close homologs can be found in several other organisms like the fungus Yarrowia lipolytica. [Martinez-Arias] [Davidow]. In biotechnology, this gene is mostly used as an auxotrophy selection marker for different yeast strains. Therefore the gene is inserted into a plasmid and into an auxotrophic yeast strain and selected for in medium without leucine (delta leu medium). For providing the iGEM community more information on the selection markers we used for our project our team we characterized the leu2 gene in different media. In our case, the leu2 gene was inserted into the F102 strain lacking his2, leu2, and ura3 genes via an integration cassette as part of the OrthoRep system. The entire cassette can be found below in the OrthoRep section. The leu2, in this case, is a part of the plasmid section which is constantly mutagenized by the orthogonal polymerase TP DNAP1. For characterization, the strain transformed with the integration cassette together with other parts of the OrthoRep system was grown in delta uracil synthetic dropout medium with different concentrations of leucine ranging from 0 mg/l to 100 mg/l. For the growth assessment, the OD at 600nm was measured every hour in a plate reader. The results are presented in the following figure. As one can see the wildtype strain is performing much better in all the media. The F102 control strain without the plasmid does not grow at all, whereas the addition of the plasmid induces growth. However, the growth of the supplemented mutagenized strain is limited compared to the wildtype. This can be linked to two known phenomena: First, the high mutagenesis rate of the leu2 containing plasmids – which is expected from the experimental setup. Second, the poor ability of auxotrophic strains to take up missing nucleotides and amino acids from the supplied medium due to altered transporter pathways, which has been reported by several groups. In the case of the studied F102 strain, these are the missing uracil and histidine which are supplemented on the synthetic media. Therefore, for optimizing experimental conditions one has to account for such growth changes in auxotrophic strains and optimize experimental conditions accordingly. This can be done by either minimizing the number of auxotrophies in the employed strain via transformation of the corresponding genes or switching to a different strain. Nevertheless, the presented experiment proves that even constantly altered this part does perform its initial function, namely substituting the missing genomic leu2 gene and providing for the growth of the auxotrophic strain in the delta leu medium. Therefore, this part can be used as a reliable marker in yeast growth assays involving complex mutagenesis screening setups in high copy plasmids such as gene library generation via OrthoRep.

improvement: BBa_K2407301

As our improvement, we present to you the Ste2 receptor integrated into the OrthoRep cassette. Ste2 originally stands for a yeast mating receptor, which is a part of the vital system for yeast sexual replication. This in its order allows for diversifying the genotype of the species. Ste2 is a transmembrane protein with a mass of around 47 kDa [Marsh1988]. In native conditions, it binds to the peptide alpha mating factor, which is 13 amino acids long. [Blumers1988] Its high sensitivity towards this small peptide as well as its wide characterization in literature makes it a suitable backbone for creating a peptide detection tool for small to middle-sized peptides. The availability of such a tool would finally close the gap that lies between the already well-established detection of large peptides and very small peptides and create new possibilities in early disease diagnostics as well as for biomonitoring and basic research. However, the mutagenesis process of such a receptor is a non-trivial task. One of the most time- consuming steps is the jump from generating a mutagenized receptor library to the actual selection of the appropriate mutants. The integration of the ste2 gene into the OrthoRep integration cassette might present an elegant solution to the described problem. As was previously described in our project proposal as well as in our best composite part page the OrthoRep system is a tool for rapid in vivo mutagenesis in yeast. The integration of the ste2 gene in the system would thus allow generating a mutant library without having to spend time and money on expensive gene block assembly and the excessive amount of additional PCRs and transformations needed to insert them into the model organisms. Further, putting a marker like GFP or his2 gene under the FUS promotor would allow for the immediate qualitative and quantitative selection of the successful mutants. The improved sequence, as well as its model, is presented below.

<groupparts>iGEM19 Ruperto_Carola</groupparts>