Team:Czech Republic/Project/Synthetic haploids

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Protocols page:

Protocols

Intro / Background

Test [Janiak2005].

Overview

Key Achievements

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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

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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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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