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
- 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
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
Relevant sequence files
- sequence of TAP tag only here
- sequence of pBS1479 here
Needed primers
Needed primers
needed to
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forward primer (5' to 3')
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reverse primer (5' to 3'
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add TAP TRP to C-term of unknown 8
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check TAP
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add TAP TRP to C-term of RNT1
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Genes to tag
Option 1: S. cerevisiae SAGA subunits
Subunit
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size,chromosome,null p-type
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Ada subunits
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Ada1 (aka HFI1, SUP110, SRM12, GAN1)
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1.467 kb=489 aa, Chr. XVI, viable
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Ada2 (aka SWI8)
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1.305 kb=434aa, Chr. IV, viable
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Ada3(aka NGG1, SWI7)
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2.109 kb=702aa, Chr. IV, viable
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Gcn5 (aka ADA4, SWI9)
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1.32 kb=439aa, Chr. VII, viable
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Ada5 (aka SPT20)
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1.815 kb=604aa, Chr. XV, viable
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Spt subunits
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Spt3
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1.014 kb=337aa, Chr. IV, viable
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Spt7(aka GIT2)
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3.999 kb=1332aa, Chr. II, viable
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Spt8
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1.809 kb=602aa, Chr. XII, viable
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Spt20 (aka Ada5)
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1.815 kb=604aa, Chr. XV, viable
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TAF subunits
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TAF5 (aka TAF90)
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2.397 kb=798aa, Chr. II, inviable
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TAF6 (aka TAF60)
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1.551 kb=516aa, Chr. VII, inviable
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TAF9 (aka TAF17)
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0.474 kb=157aa, Chr. XIII, inviable
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TAF10 (aka TAF23, TAF25)
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0.621 kb=206aa, Chr. IV, inviable
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TAF12(aka TAF61, TAF68)
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1.620 kb=539aa, Chr. IV, inviable
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Tra subunit
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Tra1
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11.235 kb=3744aa, Chr. VIII, inviable
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Other subunits
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Sgf73
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1.974 kb=657aa, Chr. VII , viable
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Sgf29
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0.779 kb=259aa, Chr. III, viable
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Sgf11
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0.3 kb=99aa, Chr.XVI, viable
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Ubp8
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1.416 kb=471aa, Chr. XIII, viable
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Sus1
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gene with intron, Chr. II, viable
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Option 2: Unknown ORFs from SGF73/sgf73 array
Unknown
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ORF
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SGF73 green signal
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sgf73 red signal
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log2 (green/red)
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1
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YHR033W
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38938 and 69586
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285 and 570
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7.1 and 6.9
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2
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YOR302W
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3374 and 6054
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49 and 167
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6.1 and 5.2
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3
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YJR097W
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524 and 1052
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13 and 28
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5.3 and 5.2
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4
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YBL028C
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1146 and 2706
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32 and 323
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5.2 and 3.1
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5
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YDR034W-B
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17290
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447
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5.3
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6
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YGR067C
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12025
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320
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5.2
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7
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YKL037W
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8340
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282
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4.9
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8
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YER067W
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6296 and 12450
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82556 and 81036
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-3.7 and -2.7
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