Difference between revisions of "Team:Czech Republic/Project/Synthetic haploids"
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==== Construction of reporter plasmids ==== | ==== Construction of reporter plasmids ==== | ||
| − | The pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome ( isolated according to standard protocol from 7283 MATx strain) | + | The pADH1, pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome (isolated according to standard protocol from 7283 MATx strain). The asCYC1 and pTv3 promoters were obtained by PCR from g-blocks. The primers used for this are as follows : |
Forward: TACTAGTAGCGGCCGCTGCAG | Forward: TACTAGTAGCGGCCGCTGCAG | ||
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'''INSERT TABLE''' | '''INSERT TABLE''' | ||
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==== Construction of INSERT MATa and INSERT MATx ==== | ==== Construction of INSERT MATa and INSERT MATx ==== | ||
Revision as of 07:05, 7 September 2015
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Protocols page:
Intro / Background
Test [Janiak2005].
Overview
Key Achievements
PUT THIS ELSEWHERE WIKI PEOPLE
- Hello
Design
Concept
Signals
TODO: Scheme of the MF(ALPHA)1 - Benchling?
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
Construction of reporter plasmids
The pADH1, pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome (isolated according to standard protocol from 7283 MATx strain). The asCYC1 and pTv3 promoters were obtained by PCR from g-blocks. The primers used for this are as follows :
Forward: TACTAGTAGCGGCCGCTGCAG Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
Forward: TACTAGTAGCGGCCGCTGCAG Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
Forward: TACTAGTAGCGGCCGCTGCAG Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
The asCYC1, and pTv3 promoters were obtained by PCR from g-blocks The primers used for this are as follows
Forward: TACTAGTAGCGGCCGCTGCAG Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
All promoters were PCRed in a single PCR run, with the following conditions . PCR products were gel verified
INSERT PHOTO_1
PCR products were then purified () and restricted by corresponding restriction enzymes, which are listed in the following table
INSERT TABLE
Construction of INSERT MATa and INSERT MATx
Validation
Results
Final constructs
Proof of concept test
Test of mating types
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