User:Nkuldell/SAGA swap pt 2

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Contents

DNA Reagents

Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [1]. Output show on SAGA swap discussion page [[2]]

BBa_Y00073 [[3]]

had DNA 2.0 codon-optimize S. pombe gene SPCC126.04c [[4]] including biobrick ends for expression of ORF in S. cerevisiae This subunit is homolog of S. cerevisiae SGF73 [[5]] subunit of SAGA and deletion of this subunit in S. cerevisiae results in viable cells but no induction of PAU genes under hypoxic conditions.

BBa_Y00029 link coming soon

Message to DNA 2.0:
The gene is an S. pombe gene called SPBC1921.07c. The protein sequence is:
mvrpinaeed vtsmwvkfhe slnpirssli kqeecyktvd gddnpieeri kacdagiqts eeqkkeleht mqslemiinv lekanekpvi tnspltrsrr nrgtsftant vtftpgmsva fklpytrhne ggdwiqciii kvtgegakqr fevqdpepdd dgnagqiykt tanhliqipa kgtplppisp ktnvlarype tttfyraevi rtlpdgsckl rfegeeevgk etvverhlvl eyng stop

What I'd like to try is to codon optimize this protein for expression in S. cerevisiae (nuclear expression, not mitochondrial as before!). Again this ORF can have none of the "forbidden" sites (EcoRI, XbaI, NotI, SpeI or PstI ) that are used for cloning. Once it's been optimized the cloning sites can be added by tagging the 5' end before the ATG with: GTTTCTTCGAATTCGCGGCCGCTTCTAGAG and tagging the 3' end after the stop with: TACTAGTAGCGGCCGCTGCAGGAAGAAAC.


Primers

  • sgf73::KanMX_KO_fwd (JW109)
    • 5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAA ctgtgcggtatttcacaccg
  • sgf73::KanMX_KO_rev (JW110)
    • 5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAA agattgtactgagagtgcac
  • sgf73_URA3_KO_fwd (NO99)
    • 5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAAatgtcgaaagctacatataa
  • sgf73_URA3_KO_rev (NO100)
    • 5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAA ttagttttgctggccgcatc
  • sgf29_URA3_KO_fwd (NO101)
    • 5' GACTTTTTCACAGCAAAACACACGGTCACCTTTCTTATTatgtcgaaagctacatataa
  • sgf29_URA3_KO_rev (NO102)
    • 5' AGAAGATCTTATGATATGTAGTAAATGTTAACCACCATTttagttttgctggccgcatc
  • SGF73_100upstm_fwd (NO107)
    • 5'- CCA GCA TCG CTC ATT AGA GAT TAA TTT
  • SGF73_100dwstm_rev (NO108)
    • 5'- GCG TAA CTT GTC CAC AAA CAT AGA CGT G
  • SGF29_100upstm_fwd (NO109)
    • 5'- CGA TTT TGC CAG ATA CGA AAA AGG TTA AAG AC
  • SGF29_100dwstm_rev (NO110)
    • 5'- CAC CTT CAA GTG AAT ACA TCA CTT CCG ACG
  • URA3_464bpDwstATG_fwd (FO3672 =MH243) Tm 58.8
    • 5'- GCAAGGGCTCCCTAGCTACT
  • URA3_638bpDwstATG_rev (FO3673 =MH244) Tm 55
    • 5'- AATGCGTCTCCCTTGTCATC
  • SpSGF73*toSc_fwd (where * indicates codon optimized version of the Sp homolog of the Sc SGF73) (NO111)
    • 5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAAatgctaaccgaacaacaaa
    • landing seq has Tm of 50°
  • SpSGF73*toSc_rev (NO112)
    • 5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAAttacagagcttgacgggct
    • landing seq has Tm of 55°
    • dangit. primer's proper sequence should be:5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAAttacagaggttgacgggct
      (ordered and logged as NO113)
      this glitch changes last 5 residues from ARQPL* to ARQAL*
  • SpSGF73toSc_fwd (to amplify Sp genomic sequence for SPCC126.04c = Sc homolog of Sc SGF73) (NO114)
    • 5' TGAACACACAAGAGAAGCGCAAAAGAGTAAAGAGCTAAAatgttaacagaacaacaga
    • landing seq has Tm of 46.3°
  • SpSGF73toSc_rev (NO115)
    • 5' CTCACTTCGTGAACATGCTGGATAACGTGCATGATTCAAttatagtggttggcgggca
    • landing seq has Tm of 56.7°
  • SpSGF29toSc_fwd (to amplify Sp genomic sequence for SPBC1921.07c = Sc homolog of Sc SGF29) (NO116)
    • 5' GACTTTTTCACAGCAAAACACACGGTCACCTTTCTTATTatggtacgcgggataaatg
    • landing seq has Tm of 52.5°
  • SpSGF29toSc_rev (NO117)
    • 5' AGAAGATCTTATGATATGTAGTAAATGTTAACCACCATTttaaccattgtactctaac
    • landing seq has Tm of 43.7°

Yeast Strains

MH176=hypoxic gene expression reporter strain

from Mark Hickman in Fred's lab:
MATa ura3D0 his3D200 leu2D0 lys2-128d HAP1 DAN3-HIS3
geneology note: = yMH36 DAN3-HIS3 #5 (targeted PAU6, went into DAN3) 05/25/05

related email note 07.12.06 from Mark
Natalie,
I only have PAU5-URA3 or DAN3-HIS3. The DAN3 reporter is tighter on plates, perhaps because the His phenotype is tighter in general. I have not checked whether SGF73 is required for PAU5-URA3 or DAN3-HIS3 induction specifically. But I do know that in an sgf73 delta mutant, there is no hypoxic PAU expression. So I infer that PAU5 and DAN3 expression is affected. We can talk in the afternoon.
Mark

FY2475 = sgf73::KanMX K/O strain

made by Jenny in Fred's lab:
MAT alpha ura3-52 lys2-173R2 leu2D1 arg4-12 his4-917d sgf73D0::KanMX

FY2474 = sgf29::KanMX K/O strain

also by Jenny?
MAT A ura3-52 lys2-173R2 leu2D1 arg4-12 his4-917d sgf73D0::KanMX

NY350 and NY351

MATa ura3D0 his3D200 leu2D0 lys2-128d HAP1 DAN3-HIS3 sgf73::URA3

  • geneology: transformed MH176 to Ura+ with product of PCR with KO primers NO99 and NO100
  • Insertion confirmed by PCR using primers that flank the K/O (NO107, NO108) as well as flank+internal PCR (NO107, NO108, FO3672)
  • Phenotype checked for failure to induce DAN3-HIS3 fusion under hypoxic conditions.
    • only NY350 grew normally on plates with ergosterol and tween.
    • This was true for SC+ erg + tween and SC-his + erg +tween, under aerob and hypoxic conditions. Use ONLY NY350!!

NY352

MAT A ura3-52 lys2-173R2 leu2D1 arg4-12 his4-917d sgf73D0::URA3 sgf29::KanMX

  • geneology: transformed FY2474 to Ura+ with product of PCR with KO primers NO99 and NO100
  • Insertion confirmed by PCR using primers that flank the K/O (NO107, NO108) as well as flank + internal PCR (NO107, NO108, FO3672)

NY356 and NY357

MAT alpha ura3-52 lys2-173R2 leu2D1 arg4-12 his4-917d sgf29::URA3 sgf73D0::KanMX

  • geneology: transformed FY2475 to Ura+ with product of PCR with KO primers NO101 and NO102 using pRS406 as template.
  • gel purified frag for transformation
  • Insertion checked by PCR using primers internal +flanking K/O (NO109, NO110, FO3672)


Phenotype MH176 (~wt) v FY2475 (sgf73::KanMX) v FY2474 (sgf29::KanMX)

First spot where individual colonies seen is listed
Better wt control would be FY3 since FY strains are all hap1

Plate type Temp MH176 (~wt) FY2475 (sgf73::KanMX) FY2474 (sgf29::KanMX) comments
YPD 30° 3d 6 6 6 Mark has seen 73 KO grow more slowly doing growth curve
YPD 37° 3d 6 6 6
YPD 14° 4d 4 4 4
YPgal 30° 3d 6 4 (repeat this?) 6
YPgal 37° 3d 5 5 5
YPgal 14° 4d 2 2 2
YPKAc 30° 3d 5 5 5
YPKAc 37° 3d 5 5 5
YPKAc 14° 4d 4 4 4
YPEG 30° 3d 3-4 2-3 2-3
YPEG 37° 3d 2-3 1-2 1-2
YPEG 30° 4d 6 3 3-4
YPEG 37° 4d 3 2 2 greater diff. seen at 30° on YPEG
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