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| − | == Intro / Background ==
| + | #REDIRECT [[Team:Czech_Republic/Project/Orthogonal_signals_and_receptors]] |
| − | Test {{:Team:Czech_Republic/Template:ReferenceRef|Janiak2005}}.
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| − | == Design ==
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| − | === Concept ===
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| − | ==== Receptors ====
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| − | ==== Signals ====
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| − | Alpha-factor mating pheromone is a short peptide (typically 13aa long) secreted by alpha cells. For our project, we needed to produce a variety of these pheromones and secrete them from the cells.
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| − | Altough there are some secretion tags in the registry (BBa_K416003, BBa_K792002), there are no parts encoding pheromones available. Also, since we found evidence that the complete wild-type signal tag is needed for the proper secretion of the pheromone {{:Team:Czech_Republic/Template:ReferenceRef|Caplan1991}}, we decided to obtain the whole sequence directly from the MF(ALPHA)1 locus.
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| − | MF(ALPHA)1 gene codes for four mature alpha-factors within a putative precursor of 165 amino acids. This sequence begins with a signal tag for secretion and a segment with three glycosylation sites (89aa). The second segment contains four mature alpha-factors, each preceded by spacer peptides, which are hypothesized to contain proteolytic processing signals{{:Team:Czech_Republic/Template:ReferenceRef|Kurjan1982}}.
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| − | === DNA ===
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| − | * Genomic PCR of WT SC-STE2 (YFL026W - http://www.yeastgenome.org/locus/S000001868/overview) - ORF
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| − | * Genomic PCR of MF(ALPHA)1 (YPL187W - http://www.yeastgenome.org/locus/mf%28alpha%291/overview) - secretion tag (first PCR), second PCR to add the actual pheromone and stop codon
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| − | == Materials and methods ==
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| − | === Chemicals and strains ===
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| − | === Construction ===
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| − | === Validation===
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| − | == Results ==
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| − | === Proof of concept test ===
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| − | === Test of orthogonality (growth arrest approach) ===
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| − | === Test of orthogonality (fluorescence approach) ===
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| − | === Final constructs ===
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| − | == References ==
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| − | <ol>
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| − | <li>{{:Team:Czech_Republic/Template:Reference|Janiak2005|Janiak, A. M., Sargsyan, H., Russo, J., Naider, F., Hauser, M., & Becker, J. M. (2005). Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae. Fungal Genetics and Biology, 42(2005), 328–338. doi:10.1016/j.fgb.2005.01.006}}</li>
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| − | <li>{{:Team:Czech_Republic/Template:Reference|Kurjan1982|Kurjan J, Herskowitz I. (1982) Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933-43. doi:10.1016/0092-8674(82)90298-7}}</li>
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| − | <li>{{:Team:Czech_Republic/Template:Reference|Caplan1991|Caplan, S., Green, R., Rocco, J., & Kurjan, J. (1991). Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway. Journal of Bacteriology, 173, 627–635. doi:10.1039/c1mb05175j}}</li>
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| − | </ol>
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