Difference between revisions of "Team:Czech Republic/Project/Orthogonal signals and receptors"
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==== Signals ==== | ==== Signals ==== | ||
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. | 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. | ||
| − | 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. | + | Altough there are some secretion tags present 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. |
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 contain proteolytic processing signals {{:Team:Czech_Republic/Template:ReferenceRef|Kurjan1982}}. | 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 contain proteolytic processing signals {{:Team:Czech_Republic/Template:ReferenceRef|Kurjan1982}}. | ||
| − | + | ||
| + | [[File:Czech_Republic_pheromone_orf.png|700px|Scheme of MF(ALPHA)1]] | ||
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Since we wanted the pheromone peptides to be easily changeable by Site-directed mutagenesis (SDM), we decided to preserve the signal tag with only one copy of the pheromone: | Since we wanted the pheromone peptides to be easily changeable by Site-directed mutagenesis (SDM), we decided to preserve the signal tag with only one copy of the pheromone: | ||
| − | + | [[File:Czech_Republic_pheromone_final_orf.png|700px|Scheme of the final ORF]] | |
| + | |||
| − | This | + | This would create a unique site for the SDM. The final production level of the pheromone is supposed to be practically the same, since the used plasmid is present in the cell in more copies (2-5). |
=== DNA === | === DNA === | ||
Revision as of 07:49, 8 September 2015
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Protocols page:
Intro / Background
Test [Janiak2005].
Design
Concept
Receptors
Alpha factor receptor (STE2) is produced and displayed by a cells. [Lin2011]
Signals
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. Altough there are some secretion tags present 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 [Caplan1991], we decided to obtain the whole sequence directly from the MF(ALPHA)1 locus.
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 contain proteolytic processing signals [Kurjan1982].
Since we wanted the pheromone peptides to be easily changeable by Site-directed mutagenesis (SDM), we decided to preserve the signal tag with only one copy of the pheromone:
This would create a unique site for the SDM. The final production level of the pheromone is supposed to be practically the same, since the used plasmid is present in the cell in more copies (2-5).
DNA
- Genomic PCR of WT SC-STE2 (YFL026W - http://www.yeastgenome.org/locus/S000001868/overview) - ORF
- 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
Materials and methods
Chemicals and strains
Construction
Validation
Results
Proof of concept test
Test of orthogonality (growth arrest approach)
Test of orthogonality (fluorescence approach)
Final constructs
References
- ↑ Lin, C.-H., Choi, a., & Bennett, R. J. (2011). Defining pheromone-receptor signaling in Candida albicans and related asexual Candida species. Molecular Biology of the Cell, 22(24), 4918–4930. doi:10.1091/mbc.E11-09-0749
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ 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