20.109:TAP-tags
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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
- 1-1 Design primers
- 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
- Mass Spec analysis description/tour/lab?
Experimental Details
Bacteria
- FB2305 = NB 311
- MC1066+pBS1539 (AmpR+URA3-Kl, TAP tag)
- FB2306 = NB312
- 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
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
Relevant sequence files
Needed primers
needed to | forward primer (5' to 3') | reverse primer (5' to 3' |
---|---|---|
add TAP TRP to C-term of unknown 8 | NO222 GGT ATT TGT CGA TTA CAA AAC ACA TCC TGT AGG CGC AAA TTC CAT GGA AAA GAG AAG 57 mer Tm = 66.7 landing Tm (17bases only) = 45.5 |
NO223 GTT AGG ATT CAG TAC TAA CAC ATT CTC TAT GAC ACA ACC TTA CGA CTC ACT ATA GGG |
check TAP | NO224 TCC ATG GAA AAG AGA AGA TGG AAA AAG AAT TTC ATA GCC GTC TC 44 mer Tm = 63.6 use with reverse primer from tagging PCR |
NO225 TCA GGT TGA CTT CCC CGC GGA ATT CGC GTC TAC 33 mer Tm = 67.9 use with fwd primer from tagging PCR expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479) |
add TAP TRP to C-term of RNT1 | NO226 CTC ACA AAA GAA TAA GAA AAG AAA ATT CTC AGA TAC AAG CTC CAT GGA AAA GAG AAG 57 mer Tm = 63.8 |
NO227 ATC AAT GCA AGT TCC ATC ATG GTT GTG TAA AAG GAA CGT TTA CGA CTC ACT ATA GGG 57 mer Tm = 66.9 |
longer landing sequences | TCC ATG GAA AAG AGA AGA TG = 20 mer fwd landing Tm = 49.6 ºC |
TAC GAC TCA CTA TAG GGC GA = 20 mer rev landing Tm = 54.3 ºC |
Unknown 8 + longer landing sequences | NO228 GT ATT TGT CGA TTA CAA AAC ACA TCC TGT AGG CGC AAA TTC CAT GGA AAA GAG AAG ATG 59 mer Tm = 66.5 ºC |
NO229 TT AGG ATT CAG TAC TAA CAC ATT CTC TAT GAC ACA ACC TTA CGA CTC ACT ATA GGG CGA 59 mer Tm = 66.6 ºC |
RNT1 + longer landing sequences | NO230 TCACAA AAGAATAAGA AAAGAAAATT CTCAGATACA AGC TCC ATG GAA AAG AGA AGA TG 59 mer Tm = 64.4 ºC |
NO231 TCA ATG CAA GTT CCA TCA TGG TTG TGT AAA AGG AAC GTT TAC GAC TCA CTA TAG GGC GA 59 mer Tm = 68.4 ºC |
check TAP | NO258 for SPT3-TAP control TTT AAA GGT GGT AGA CTC AGT TCT AAA CCA ATT ATC ATG tcc atg gaa aag aga aga tg 59 mer use with NO225 = reverse primer for checking TAP tag expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479) |
NO259for SPT7-TAP control AAT AGT TCA TTT AGC TTG AGC CTT CCT CGC CTT AAT CAA tcc atg gaa aag aga aga tg 59 mer use with NO225 = reverse primer for checking TAP tag expect length of TAP seq + TRP K. l seq = 554 bps (from 2147 to 2701 in pBS1479) |
Check TAP with NO224 | SPT3_check_rev NO268 ACC AGA AGG AAA CCC ATG CAC CTC CAT GAT GAA ATT ATA CAA AAA T |
SPT7_check_rev NO269 AGC AAA TGT ATT TAA TTA AAG ATA AAA TAT TCA ACT ATT TAG CGC GCT CA |
Genes to tag
Option 1: S. cerevisiae SAGA subunits
Subunit | size,chromosome,null p-type |
---|---|
Ada subunits | |
Ada1 (aka HFI1, SUP110, SRM12, GAN1) | 1.467 kb=489 aa, Chr. XVI, viable |
Ada2 (aka SWI8) | 1.305 kb=434aa, Chr. IV, viable |
Ada3(aka NGG1, SWI7) | 2.109 kb=702aa, Chr. IV, viable |
Gcn5 (aka ADA4, SWI9) | 1.32 kb=439aa, Chr. VII, viable |
Ada5 (aka SPT20) | 1.815 kb=604aa, Chr. XV, viable |
Spt subunits | |
Spt3 | 1.014 kb=337aa, Chr. IV, viable |
Spt7(aka GIT2) | 3.999 kb=1332aa, Chr. II, viable |
Spt8 | 1.809 kb=602aa, Chr. XII, viable |
Spt20 (aka Ada5) | 1.815 kb=604aa, Chr. XV, viable |
TAF subunits | |
TAF5 (aka TAF90) | 2.397 kb=798aa, Chr. II, inviable |
TAF6 (aka TAF60) | 1.551 kb=516aa, Chr. VII, inviable |
TAF9 (aka TAF17) | 0.474 kb=157aa, Chr. XIII, inviable |
TAF10 (aka TAF23, TAF25) | 0.621 kb=206aa, Chr. IV, inviable |
TAF12(aka TAF61, TAF68) | 1.620 kb=539aa, Chr. IV, inviable |
Tra subunit | |
Tra1 | 11.235 kb=3744aa, Chr. VIII, inviable |
Other subunits | |
Sgf73 | 1.974 kb=657aa, Chr. VII , viable |
Sgf29 | 0.779 kb=259aa, Chr. III, viable |
Sgf11 | 0.3 kb=99aa, Chr.XVI, viable |
Ubp8 | 1.416 kb=471aa, Chr. XIII, viable |
Sus1 | gene with intron, Chr. II, viable |
Option 2: Unknown ORFs from SGF73/sgf73 array
Unknown | ORF | SGF73 green signal | sgf73 red signal | log2 (green/red) |
---|---|---|---|---|
1 | YHR033W | 38938 and 69586 | 285 and 570 | 7.1 and 6.9 |
2 | YOR302W | 3374 and 6054 | 49 and 167 | 6.1 and 5.2 |
3 | YJR097W | 524 and 1052 | 13 and 28 | 5.3 and 5.2 |
4 | YBL028C | 1146 and 2706 | 32 and 323 | 5.2 and 3.1 |
5 | YDR034W-B | 17290 | 447 | 5.3 |
6 | YGR067C | 12025 | 320 | 5.2 |
7 | YKL037W | 8340 | 282 | 4.9 |
8 | YER067W | 6296 and 12450 | 82556 and 81036 | -3.7 and -2.7 |