Difference between revisions of "Team:Czech Republic/Project/Signal transduction/Matalpha"

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=MATa integration plasmid=
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=MATα integration plasmid=
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MATα integration plasmid carries a synthetic MATα locus that integrates into the wild-type locus putting both α1 and α2 transcription factors on synthetic promoters and inserting STE12 gene with its promoter.
  
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MATα integration plasmid carries a synthetic MATa locus that integrates into the wild-type locus inserting TetR and STE12 with their promoters into the center of the a1 ORF and thus disrupting it.
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Wild-type MATα locus carries genes that code for α1 and α2 transcription factors. The locus codes for α1 in one direction and for α2 in the opposite direction simultaneously. Therefore any changes in α1 gene’s promoter would disrupt the promoter of α2. This problem was resolved by putting also the α2 on a synthetic promoter.  
  
 
==Integrated parts==
 
==Integrated parts==
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All part are cloned into pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes SnabI restriction site for linearization before chromosomal integration. The whole plasmid is integrated within the a1 ORF.
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All part are cloned into pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes SnabI restriction site for linearization before chromosomal integration. The whole plasmid is integrated between the α1 and α2 ORFs.
  
  
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*TetR on ADH1 promoter
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*α1 on pTet promoter:ORF of α1 is a genomic sequence, it also serves as a homologous part for the plasmid integration. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html here] (the T16 version). This promoter is repressed by TetR (tetracycline) and is active in absence of TetR (see [https://en.wikipedia.org/wiki/Tetracycline-controlled_transcriptional_activation this wikipeadia page] for more information about tetracycline-controlled transcriptional activation).  
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Tetracycline repressor ORF sequence was taken from [https://www.addgene.org/vector-database/2525/ Addgene databese], the ADH1 promoter sequence was taken from [https://www.addgene.org/15970/ same page]. CYC1 terminator is included after TetR, it’s sequence was also taken from [https://www.addgene.org/15970/ this page].
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*α2 on CYC1 promoter:ORF of α2 is a genomic sequence, it also serves as the second homologous part for the plasmid integration. Promoter CYC1 has sequence taken from [http://www.nature.com/ncomms/2014/140527/ncomms5002/full/ncomms5002.html(the CYC1v3 version) this page].
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Show sequence
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For both α1 and α2, no synthetic terminators were designed because the terminators (native)will be already within the chromosome after integration.
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*STE12 on pTet promoter:ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Therefore, no synthetic terminator was included. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html(the T15 version) here].
  
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*STE12 on a-specific CYC1
 
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ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Sequence of a-specific CYC1 promoter was obtained from Prof. Vershon, who presented series of a-specific promoters{{:Team:Czech_Republic/Template:ReferenceRef|Vershon1999}}. It is a CYC1 promoter with inserted α2-Mcm1 binding sequence from AGA2 gene (α2-Mcm1 complex represses a-sg).
 
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Show sequence
 
  
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*Parts of a1 ORF
 
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Two parts of a1 OFR are included as homologous parts for chromosomal integration. The plasmid integrates in the center of a1 ORF and disrupts the gene.
 
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Show sequence
 
  
 
==References==
 
==References==

Revision as of 12:27, 7 September 2015

{{{1}}}

MATα integration plasmid

MATα integration plasmid carries a synthetic MATα locus that integrates into the wild-type locus putting both α1 and α2 transcription factors on synthetic promoters and inserting STE12 gene with its promoter.

Wild-type MATα locus carries genes that code for α1 and α2 transcription factors. The locus codes for α1 in one direction and for α2 in the opposite direction simultaneously. Therefore any changes in α1 gene’s promoter would disrupt the promoter of α2. This problem was resolved by putting also the α2 on a synthetic promoter.

Integrated parts

All part are cloned into pRSII406 integrating vector (from Addgene). The final plasmid shown in the picture includes SnabI restriction site for linearization before chromosomal integration. The whole plasmid is integrated between the α1 and α2 ORFs.


  • α1 on pTet promoter:ORF of α1 is a genomic sequence, it also serves as a homologous part for the plasmid integration. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html here] (the T16 version). This promoter is repressed by TetR (tetracycline) and is active in absence of TetR (see this wikipeadia page for more information about tetracycline-controlled transcriptional activation).
  • α2 on CYC1 promoter:ORF of α2 is a genomic sequence, it also serves as the second homologous part for the plasmid integration. Promoter CYC1 has sequence taken from [http://www.nature.com/ncomms/2014/140527/ncomms5002/full/ncomms5002.html(the CYC1v3 version) this page].

For both α1 and α2, no synthetic terminators were designed because the terminators (native)will be already within the chromosome after integration.

  • STE12 on pTet promoter:ORF of ST12 is a genomic sequence that was extracted from chromosome also with the 3’UTR. Therefore, no synthetic terminator was included. Promoter pTet has sequence taken from [http://www.nature.com/nbt/journal/v27/n5/abs/nbt.1536.html(the T15 version) here].


References

  1. ↑ Hualin Zhong, Ron McCord, and Andrew K. Vershon. Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome. doi: 10.1101/gr.9.11.1040 Genome Res. 1999. 9: 1040-1047 Cold Spring Harbor Laboratory Press