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

Bacteria

 * FB2305 = NB 311
 * MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
 * FB2306 = NB312
 * 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
 * NB318
 * XL1-bl+pBS1479 (Lisa's prep of plasmid which does give PCR product for TAP-TRP, freshly transformed)

Yeast

 * FY631 = Parental for TAP tagging = NY411
 * MATa, his4-917d, lys2-173R2, leu2d1, ura3-52, trp1d63
 * FY2714 = NY412
 * MATa, HA-SPT3-TAP::TRP1, his4-917d, lys2-173R2, ura3D0 or -52, leu2D1 or D0, trp1D63
 * FY2031 = NY413
 * MATa, HA-SPT7-TAP::TRP1, his4-917d, lys2-173R2, ura3D0, leu2D1, trp1D63
 * FY2305 = NY414
 * MATa, SPT10-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
 *  not his4-912d
 * FY2306 = NY415
 * MATa, SPT21-TAP::TRP1 trp1D63 his3D200 leu2D1 ura3-52 lys2-128d
 *  not his4-912d

General format for

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

Relevant sequence files

 * 1) sequence of TAP tag only [[Media:Seq TAPtag.rtf| here]]
 * 2) sequence of pBS1479 [[Media:Seq pBS1479 TAP+TRP.rtf| here]]