Difference between revisions of "Team:Ruperto Carola/Parts"
| (22 intermediate revisions by the same user not shown) | |||
| Line 26: | Line 26: | ||
</div> | </div> | ||
| − | <div class="container mt-5"> | + | <div class="container-fluid mt-5"> |
<div class="row pb-5 justify-content-center"> | <div class="row pb-5 justify-content-center"> | ||
| Line 33: | Line 33: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-12 col-md-3 pb-3 pb-md-0"> | <div class="col-12 col-md-3 pb-3 pb-md-0"> | ||
| − | <img class="img-fluid mx-auto d-block" src="https://static.igem.org/mediawiki/2019/e/ef/T--Ruperto_Carola--dice1.png" alt=""> | + | <img class="img-fluid mx-auto d-block pb-3" src="https://static.igem.org/mediawiki/2019/e/ef/T--Ruperto_Carola--dice1.png" alt=""> |
</div> | </div> | ||
| − | <div class="col-12 col-md-9"> | + | <div class="col-12 col-md-9 d-flex justify-content-center align-items-center"> |
<h2>Best basic: polymerase BBa_K3173000</h2> | <h2>Best basic: polymerase BBa_K3173000</h2> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row mt-2"> | ||
| + | <div class="col-12"> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
This part (terminal protein DNAP1) represents a DNA polymerase which was extracted from the K. | 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 <x-ref>Bergquist2002</x-ref>. This strain is also known to harbor and | + | lactis – a yeast strain actively used in biotechnology (especially fermentation) and science [<x-ref>Bergquist2002</x-ref>]. This strain is also known to harbor and |
express a linear cytoplasmic yeast plasmids. | express a linear cytoplasmic yeast plasmids. | ||
</p> | </p> | ||
| Line 49: | Line 53: | ||
Since the common yeast strains employed in research (S. cerevisiae) are lacking linear cytoplasmic | 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 | 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 <x- | + | OrthoRep system allowed us to orthogonally mutagenize genes of interest in vivo [<x-ref>Ravikumar2018</x-ref>]. |
| − | ref>Ravikumar2018</x-ref> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</p> | </p> | ||
</div> | </div> | ||
| Line 70: | Line 64: | ||
<div class="col-10 rounded shadow"> | <div class="col-10 rounded shadow"> | ||
<div class="card-body"> | <div class="card-body"> | ||
| − | + | <img class="w-25 mr-1 float-right pl-3" src="https://static.igem.org/mediawiki/2019/1/14/T--Ruperto_Carola--img-structure_swiss.png" alt=""> | |
| − | + | <p class="text-justify"> | |
| − | + | 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. | ||
| + | </p> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 79: | Line 80: | ||
<div class="col-10 rounded shadow"> | <div class="col-10 rounded shadow"> | ||
<div class="card-body"> | <div class="card-body"> | ||
| + | <img class="w-25 float-left pr-3" src="https://static.igem.org/mediawiki/2019/2/25/T--Ruperto_Carola--img-tpdnap.png" alt=""> | ||
<p class="text-justify"> | <p class="text-justify"> | ||
| Line 97: | Line 99: | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<div class="row pb-5 justify-content-center"> | <div class="row pb-5 justify-content-center"> | ||
| Line 133: | Line 127: | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| Line 382: | Line 139: | ||
Ste2 originally stands for a yeast mating receptor, which is a part of the vital system for yeast sexual | 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. | 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 | + | Ste2 is a transmembrane protein with a mass of around 47 kDa [<x-ref>Marsh1988</x-ref>]. In native |
| − | conditions, it binds to the peptide alpha mating factor, which is 13 amino acids long. | + | conditions, it binds to the peptide alpha mating factor, which is 13 amino acids long. |
| − | + | [<x-ref>Blumers1988</x-ref>] Its high sensitivity towards this small peptide as well as its wide | |
| − | ref | + | |
characterization in literature makes it a suitable backbone for creating a peptide detection tool for | 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 | small to middle-sized peptides. The availability of such a tool would finally close the gap that lies | ||
Latest revision as of 01:56, 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 [
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
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).
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 [
