Difference between revisions of "Team:Czech Republic/Project/Synthetic haploids"

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=== Construction ===
 
=== Construction ===
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==== Construction of reporter plasmids ====
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The pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome ( isolated according to standard protocol from 7283 MATx strain), The primers used for this are as follows 
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 +
Forward: TACTAGTAGCGGCCGCTGCAG
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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 
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 +
Forward: TACTAGTAGCGGCCGCTGCAG
 +
Reverse: GCTAGCCCAAAAAAACGGGTATGGAG
 +
 +
All promoters were PCRed in a single PCR run, with the following conditions . PCR products were gel verified
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'''INSERT PHOTO_1'''
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PCR products were then purified () and restricted by corresponding restriction enzymes, which are listed in the following table
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'''INSERT TABLE'''
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==== Construction of INSERT MATa and INSERT MATx ====
  
 
=== Validation===
 
=== Validation===

Revision as of 14:30, 6 September 2015

{{{1}}}


Protocols page:

Protocols

Intro / Background

Test [Janiak2005].

Overview

Key Achievements

PUT THIS ELSEWHERE WIKI PEOPLE

  1. 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 pSTE2, pSTE5, and pFUS1 promoters were obtained by PCR from yeast genome ( isolated according to standard protocol from 7283 MATx strain), 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

  1. ↑ 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