Difference between revisions of "Team:Czech Republic/Project/Communication module"

Revision as of 18:55, 30 August 2015

Intro / Background

Test ^[Janiak2005].

Design

Concept

Receptors

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 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].

TODO: Scheme of the MF(ALPHA)1 - Benchling?

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:

TODO: Final scheme of the ORF - Benchling?

This created 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

↑ 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
↑ 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

@@ Line 13: / Line 13: @@
 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.
-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}}.
+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}}.
+TODO: Scheme of the MF(ALPHA)1 - Benchling?
+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:
+TODO: Final scheme of the ORF - Benchling?
+This created 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 ===