20.109:TAP-tags

New Protein Engineering Module for F08

Point of departure is to ask if tags are actually neutral wrt protein function

Rough sketch of labs

• Part 1: TAP tag
  • non-essential SAGA subunits (essential likely in many complexes so purif profile complex + tag has many consequences)
  • unknown ORFs changed during microarray of SAGA del or mtRNT1
    • 1-1 Design primers
      
    • 1-2 PCR
      
    • 1-3 Transform
      
    • 1-4 Confirm integration by colony PCR
      
  • Alternatively, could just give students tagged version.
• Part 2: Western
  • need + TAP control
  • use Sigma Ab to protA (anti-ProtA raised in rabbit or if need to detect tag when protA has been cleaved can use OpenBiosystems Ab)
  • 1.5 ml ON culture, vortex 3X30' with glass beads and add 100 ul LB from Methods (2001) 24:218 barton link
• Part 3: Phenotypes
  • compare parental, K/O, TAP-tagged
  • examine GAL, Spt (his4-917d), TS, CS
• Part 4: Array
  • compare parental to TAP-tagged
• Could also include
  • Mass Spec analysis description/tour/lab?
    
  • Tour of protein purif facility

Experimental Details

Bacteria

• FB2305
  • MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
• FB2306
  • MC1066+pBS1479(AmpR+TRP1-Kl, TAP tag)
  • ref for seq of TRP1 from Kluyveromyces lactis here
  • NCBI link for seq of TRP1 from Kluyveromyces lactis here

from Methods 2001 article[1]

Yeast

• FY631 = Parental for TAP tagging
  • MATa, his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63
• FY2714
  • MATa, HA-SPT3-TAP::TRP1, his4-917d, lys2-173R2, ura3D0 or -52, leu2D1 or D0, trp1D63
• FY2031
  • MATa, HA-SPT7-TAP::TRP1, his4-917d, lys2-173R2, ura3D0, leu2D1, trp1D63
• FY2305
  • MATa, SPT10-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
  • not his4-912d
• FY2306
  • MATa, SPT21-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
  • not his4-912d

Primers for C-terminal fusions

General format for

• Upstream primer: 40nt then TCC ATG GAA AAG AGA AG
• Downstream primer: 40nt then TAC GAC TCA CTA TAG GG

Need primers for

• initial amplification to transform
• verification of insertion
• mtRNT1 expression