Team:Czech Republic/Project/Synthetic haploids

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Protocols page:

Protocols

Introduction:Yeast pheromone pathway

Yeast Saccharomyces cerevisiae exists either in haploid or diploid state. The two mating types are called MATa and MATα that differ only within an approx. 2 kbp long region on chromosome III called MAT locus. MATa locus expresses transcription factors a1 and a2, MATα locus expresses transcription factors α1 and α2. Both types express three groups

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of genes, which are:

  • haploid-specific genes (h-sg)
  • a-specific genes (a-sg)
  • α-specific genes (α-sg).

As their names indicate, these genes are only active in haploids, MATa cells, or MATα cells, respectively.

Both mating types constantly produce small amounts of mating type-specific pheromone and when the two cells of opposite types are in close proximity they identify each other by sensing each other’s pheromone. The response on detected pheromone is enabled through the pheromone pathway, which is a cascade of chemical reactions that results in preparation for the process of mating. Since only haploids mate, the yeast pheromone pathway is only functional in haploid cells.


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

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

The backbone pRSII406 was obtained from Addgene. MATa was ordered in the form of gblock yG_MATa with sequence

AATTCATCTAGAGAAGAAAGCAAAGCCTTAATTCCAAGGAAAAAGAAGAAGTTGCAAAGAAATGTGGCATTACTCCACTTCAAGTAAGAGTTTGGGTATGTAATATGAGAATCAAACTTAAATATATCCTATACGTAGTATGGCGGAAAACATAAACAGAACTCTGTTTAACATTCTAGGTACTGAGcaaattaaagccttcgagcgtcccaaaaccttctcaagcaaggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttcttaatactaacataactataaaaaaataaatagggacctagacttcaggttgtctaactccttccttttcggttagagcggatgtggggggagggcgtgaatgtaagcgtgacataactaatCTAAAATTCCCGGGATCCGCTGTACGCGGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTGCGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATTTTAATAAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGCAGAAAAAGAAACAAGGAAGAAAGGGAACGAGAACAATGACGAGGAAACAAAAGATTAATAATTGCAGGTCTATTTATACTTGATAGCAAGACAGCAAACTTTTTTTTATTTCAAATTCAAGTAACTGGAAGGAAGGCCGTATACCGTTGCTCATTAGAGAGTAGTGTGCGTGAATGAAGGAAGGAAAAAGTTTCGTGTGCTTCGAGATACCCCTCATCAGCTCTGGAACAACGACATCTGTTGGTGCTGTCTTTGTCGTTAATTTTTTCCTTTAGTGTCTTCCATCATTTTTTTGTCATTGCGGATATGGTGAGACAACAACGGGGGAGAGAGAAAAGAAAAAAAAAGAAAAGAAGTTGtaaacccacaccgggtgtcataatcaaccaatcgtaacttcatctcttccacccatgtctctttgagcaataaagccgataacaaaatctttgtcgctcttcgcaatgtcaacagtacccttagtatattctccagtagatagggagcccttgcatgacaattctgctaacatcaaaaggcctctaggttcctttgttacttcttctgccgcctgcttcaaaccgctaacaatacctggTccACTAGTCCCGGGAGCAAGATCAAGATGTTTTCACCGATCTTTCCGGTCTCTTTGGCCGGGGTTTACGGACGATGGCAGAAGACCAAAGCGCCAGTTCATTTGGCGAGCGTTGGTTGGTGGATCAAGCCCACGCGTAGGCAATCCTCGCAGATCTCGAACCATGTAATTTCCGAATACGGTAATTACACGCATCGAGCAGATCCGCCAGGCGTGTATATATAGCGTGGATGGCCAGGCAACTTTAGTGCTGACACATACAGGCATATATATATGTGTGCGACGACACATGATCATATGGCATGCATGTGCTCTGTATGTATATAAAACTCTTGTTTTCTTCTTTTCTCTAAATATTCTTTCCTTATACATTAGGACCTTTGCAGCATAAATTACTATACTTCTATAGACACACAAACACAAATACACACACTAAAaagctt

MATx was ordered in the form of two g-blocks, yG_MATx1 and yG_MATx2

yG_MATx1 :

AAGCTTGGATTCTCACAATCCTGTCGGTCACTTCTCGGCTGTTCGCGTATATTTTTTGTTGATACTTTTACCGGTATTTTGTCTGTAATTTATTCTCTATCACTGATAGGGACTTCTCTATCACTGATAGGGAACCCAGCCTGATTTATACTATTAGGGATCGCAGGAAGGCGGTGGGAAGTCCGGGAGTCGCTGAGGGGAAGTGTCAGTGGTTTTGGGTATAAATGGCTGGTTGTTCCCTATCAGTAATAGAGAATTCCCTATCAGTGATAGAGACTGCGGATTTAGAAACTACCTGATAAAAGTATCAACAAAAATTGCGCATGCCGGCCTGGATTTTGCGCAAATTTACCTTAACGTCCCACAATATGTTTACTTCGAAGCCTGCTTTCAAAATTAAGAACAAAGCATCCAAATCATACAGAAACACAGCGGTTTCAAAAAAGCTGAAAGAAAAACGTCTAGCTGAGCATGTGAGGCCAAGCTGCTTCAATATTATTCGACCACTCAAGAAAGATATCCAGATTCCTGTTCCTTCCTCTCGATTTTTAAATAAAATCCAAATTCACAGGATAGCGTCTGGAAGTCAAAATACTCAGTTTCGACAGTTCAATAAGACATCTATAAAATCTTCAAAGAAATATTTAAACTCATTTATGGCTTTTAGAGCATATTACTCACAGTTTGGCTCCGGTGTAAAACAAAATGTCTTGTCTTCTCTGCTCGCTGAAGAATGGCACGCGGACAAAATGCAGCACGGAATATGGGACTACTTCGCGCAACAGTATAATTTTATAAACCCTGGTTTTGGTTTTGTAGAGTGGTTGACGAATAATTATGCTGAAGTACGTGGTGACGGATATTGGGAAGATGTGTTTGTACATTTGGCCTTATAGAGTGTGGTCGTGGCGGAGGTTGTTTATCTTTCGAGTACTGAATGTTGTCAGTATAGCTATCCTATTTGAAACTCCCCATCGTCTTGCTGCAG

yG_MATx2 :

CTGCAGAGTAGTGTCTGAGGTACAAACATCTTAGTAGTGTCGAGAGGGTTGATTGTTTATGTATTTTTGCGAAATATATATATATATATTCTACACAGATATATACATATTTGTTTTTCGGGCTCATTCTTTCTTCTTTGCCAGAGGCTCACCGCTCAAGAGGTCCGCTAATTCTGGAGCGATTGTTATTGTTTTTTCTTTTCTTCTTCTATTCGAAACCCAGTTTTTGATTTGAATGCGAGATAAACTGGTATTCTTCATTAGATTCTCTAGGCCCTTGGTATCTAGATATGGGTTCTCGATGTTCTTTGCAAACCAACTTTCTAGTATTCGGACATTTTCTTTTGTAAACCGGTGTCCTCTGTAAGGTTTAGTACTTTTGTTTATCATATCTTGAGTTACCACATTAAATACCAACCCATCCGCCGATTTATTTTTCTGTGTAAGTTGATAATTACTTCTATCGTTTTCTATGCTGCGCATTTCTTTGAGTAATACAGTAATGGTAGTAGTGAGTTGAGATGTTGTTTGCAACAACTTCTTCTCCTCATCACTAATCTTACGGTTTTTGTTGGCCCTAGATAAGAATCCTAATATATCCCTTAATTCAACTTCTTCTTCTGTTGTTACACTCTCTGGTAACTTAGGTAAATTACAGCAAATAGAAAAGAGCTTTTTATTTATGTCTAGTATGCTGGATTTAAACTCATCTGTGATTTGTGGATTTAAAAGGTCTTTAATGGGTATTTTATTCATTTTTTCTTAGTGTGTGTATTTGTATTTGCGTGTCTATAGAAGTATAGTAATTTATGCTGCAAAGGTCCTAATGTATAAGGAAAAAAAATTTAGAGAAAAAAAGAAAAAAAGAGTTTTATATACATACAGAGCACATACATGCCATATAATCATGTATATACGCGCACATATATATATGCCTGTATGTGTCAGCACTAAATTTACCTGAACATACGCGCTATATATACGCGCCTCGCGTATATGCTCGAGGATTCCCTACGCGTGGGCTTTTTTTACTAACCAACGCGCGCGAAATActagt

The g-blocks were restricted by the following enzymes, along with the vector

insert table 1

Restriction were purified (insert purification kit name) and ligated together using the standard ligation protocol. STE12 was PCR amplified from yeast genome (isolated from 7283 MATx strain) using the following primers : Forward : CTTGTAAAGCTTCCAAGGATGAAAGTCCAAATAACCAATAGTAGAACA Reverse : ACTGCACTCGAGAGATTTGTTACATTTATTACCTTTTTTTCTTGCTTT The conditions for the PCR were the following

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