|TR||Tn3 terminal inverted repeats|
|HA||Hemagglutinin (HA) epitope |
|loxR||lox site, target for Cre recombinase|
|lacZ||5'-truncated lacZ gene encoding beta-galactosidase|
|URA3||URA3 gene from S. cerevisiae|
|tet||Tetracycline resistance gene|
|res||Tn3 site for resolution of transposition intermediate|
|loxP||lox site, target for Cre recombinase|
|3xHA||Hemagglutinin (HA) triple epitope tag|
Uses: Gene disruption, analysis of gene expression, HAT epitope-tagging protein at range of sites, creating conditional alleles.
In more detail:mTn-4xHA/lacZ can easily be inserted at mutiple sites in a given gene. The mutagenized DNA is then transformed into yeast, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. Insertions that generate in-frame fusion of the coding region to lacZ can be used to monitor and quantify gene expression, via assays for beta-gal activity. The transposon can also be excized by Cre-mediated recombination to leave a 5 base-pair duplication caused by transposon insertion plus a 262-bp insertion containing sequences encoding 4 copies of the HA epitope. When lacZ is fused in-frame to the gene of interest, the excision event results in an in-frame insertion of 89 amino acids, called a HAT tag, into the encoded protein. Insertion of the HAT tag has the potential to create conditionally-defective forms of the protein.
The accession for mTn-4xHA/lacZ is U54829.
A kit for mutagenesis of a yeast gene with mTn-4xHA/lacZ is available.
Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-4xHA/lacZ
Please read this whole document before you start!
- Clone fragment into vector pHSS6.
- pHSS6 is from strain R1123; map given below.
- Delete as much of the polylinker as possible as sometimes transposon 'hot-spots' into it.
- Select transformants on LB Kan40.
- Transform this plasmid into competent cells of R1236/B211.
- Transfer F::mTn-4xHA/lacZ into cells by mating with strain #94.
- Grow strains overnight with antibiotic selection (Tet3 for #94).
- Subculture 1:100 in fresh medium (no antibiotics). Grow at 37oC to early log phase (when cell swirls are visible). The recipient strain (B211) can be denser than the donor.
- Mix 200 ul of each strain. Incubate at 37oC without agitation for 20 min to 1 hr.
- Plate as 100 ul aliquots onto LB Tet3 Kan40 Cm34.
- Do the Control: Spot the starting strains onto this media.
- Grow 1-2 days at 30oC. Now you have cointegrates.
- Set up strain #70 in Sm50 Cm34 overnight.
- Mate to strain #70 to resolve cointegrates
- Elute colonies from plates: put 2 mls of LB on the plate, scrape off the colonies with a speader. This is your eluate. You should have several thousand colonies at least.
- Dilute overnight culture of strain #70 1:100 without antibiotic. Dilute eluate to roughly same density.
- Grow and mate as before.
- After mating for 20 min to 1 hr, plate 100 ul aliquots on LB Tet3 Kan40 Sm50 and grow overnight at 37oC.
- Do the Control: Spot the starting strains onto this media.
- Rescue resolved DNA from this strain
- Elute your colonies off in LB. Again, you should have thousands. Dilute some eluate in LB Tet3 Kan40 to give an almost saturated density. Grow at 37oC for a few hours.
- Isolate DNA by miniprep. (We do a standard 1-2-3 alkaline lysis but use 150 ul of 7.5M NH4Ac as solution III, and 270 ul of isopropanol to precipitate. This removes most of protein (avoiding phenol) and RNA, giving a very small clean pellet. Still, there are nucleases so we keep everything on ice).
- Transform about 1/10 of minprep into a regular recA endAcloning strain (eg DH5). Plate on LB Tet3 Kan40.
- Transform into yeast.
- Elute entire pool of transformants (again, aim for thousands) and make a miniprep as in step 5. (Make -70oC stock of bacterial pool for future use).
- Transform NotI digest of entire pool into yeast, selecting for URA3. Look for lacZ fusions among transformants.
- N.B.For HAT epitope-tagging, you may want to pre-transform your yeast strain with pB227/GAL-cre (selecting LEU2).
Screening for in-frame lacZ fusions in yeast
- Transformant colonies are patched to SC-ura ( SC-ura -leu if you already have pB227/GAL-cre in there).
- Cells are replica plated to an SC-ura (-leu) plate and a SC-ura plate on which a sterile disc of Whatman 1A filter paper has been placed, and grown overnight at 30oC. Other media or growth conditions can be substituted as desired. For ade2 strains, any test media should contain 80 mg/l of adenine, as the red pigment can obscure the X-gal result.
- Filters are lifted from the plates and placed in the lid of a 9-cm glass petri dish. This lid is then placed inside a closed 15-cm glass petri dish containing chloroform, for 10 to 30 minutes. The minimum exposure time necessary for a particular yeast strain can be determined empirically.
- Filters are placed colony-side up onto X-Gal plates (120 ug/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, 0.1 M NaPO4 [pH 7] and 1 mM MgSO4 in 1.6% agar) and incubated at 30oC for up to 2 days.
- Transformants carrying productive lacZ fusions are recovered from the regrown SC-ura (-leu) plate . It is advisable to subsequently maintain selection for URA3 wherever possible, as some mutations are deleterious even in the heterozygous state.
- PCR primers designed using the mTn-3xHA/lacZ sequence can be used to determine position of the transposon. The IR elements and palindromic lox regions should be avoided.
Using the excision feature to HAT-epitope tag a protein
A leu2 ura3 GAL+ yeast strain is required. When transposon insertion has created an in-frame fusion to lacZ in the gene of interest, the transposon can be excized by Cre-mediated recombination to leave a 262-bp insertion (sequence given below) containing four copies of the HA epitope. With the 5 base pair duplication caused by transposon insertion, this gives an in-frame 89 amino acid insertion. The popout event is mediated by cre recombinase and requires induction of the GAL1-10 promoter on galactose. Our strains grow poorly on galactose but give 80 to 100% popouts.
The HA tag can be detected by mouse monoclonal antibodies 12CA5 (Boehringer) or MMS101R (BAbCo, Richmond, California). These antibody recognise cross-reacting yeast proteins of about 55kD or110kD, respectively, and can give a spotty background on immunofluorescence. Despite this drawback, the 3xHA tag has been used extensively and successfully in yeast. A rabbit polyclonal antisera is also available (101c500; BabCo) but this was less reactive in the one instance we tried. Protocols for yeast immunofluorescence can be found here, or in Methods in Enzymology 194 (1991).
- Transform strain with plasmid pB227/GAL-cre, selecting on SC-leu.
- Inoculate transformants into 2mls SC-ura-leu with 2% raffinose as carbon source, and grow to saturation.
- Dilute 1/100 into SC-leu with 2% galactose as carbon source. As a control also dilute 1/100 into SC-leu with 2% glucose as carbon source. Grow for 2 days (some strains induce without growing).
- If grown, dilute 1/100. Otherwise, proceed with undiluted culture.
- Spot a 10ul drop onto an FOA plate and streak it for single colonies (non-quantitative approach!).
- Alternatively, plate dilutions onto SC media and replica to SC-ura to identify Ura- colonies. The induced cultures should give 100-fold more Ura- cells than the control.
- PCR primers designed using the sequence given below can be used to determine position of the tag. The TR elements and palindromic loxR region should be avoided.
N.B. When tagging essential genes, the original strain transformed should obviously be diploid. You can dissect the HAT-tagged version to see if the tagged gene is functional. To be rigourous, only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.
Sequence of HAT tag (4xHA):
TR in upper case. loxR in bold.
GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggcctaccca tacgacgtcc cagactacgc gttggccgct atcgcttcgg ataactcctg ctatacgaag ttatgggcgg ccgtttaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg caggatccta tccatatgac gttccagatt acgctccggc cgcCCTTAAC GTGAGTTTTC GTTCCACTGA GCGTCAGACC CC
|R1123||Strain XL1-blue carrying vector pHSS6|
|R1236/B211 ||Strain RDP146 (F- recA' (deletion lac-pro) rpsE; spectinomycin resistant) with plasmid pLB101 (pACYC184 with tnpA; active transposase, chloramphenicol resistant)(F. Heffron)|
|#94 /B426||Strain RDP146 with pOX38 F factor derivative carrying mTn3 derivative mTn-4xHA/lacZ (lacZ, URA3, tet; tetracycline resistant)|
|#70/B425||Strain NG135 (K12 recA56 gal-delS165 strA; streptomycin resistant) with plasmid pNG54 (pACYC184 with mTn3 res and tnpR seqs; active resolvase, chloramphenicol resistant)(N. Grindley)|
|B227||Strain DH5-alpha carrying pB227/GAL-cre (amp, ori, CEN, LEU2) (B. Sauer)|
The accession for pHSS6 is M84115.
|Tetracycline, Tet (Sigma T3383)||12 mg/ ml in 50% ethanol. Use at 3 ug/ml (Tet3)|
|Kanamycin, Kan (Sigma K800) ||10 mg/ ml in water. Use at 40 ug/ml (Kan40)|
|Chloramphenicol, Cm (Sigma C0378, I think)||34 mg/ml in ethanol. Use at 34 ug/ml (Cm34)|
|Streptomycin, Sm (Sigma S6501)||10 mg/ml in water. Use at 50 ug/ml (Sm50)|
|Ampicillin, Amp (Sigma A9518)||50 mg/ml in water. Use at 50 ug/ml (Amp50)|
When only a few plates of each type are used, it's convenient to chop an LB plate up with a sterile toothpick, put the bits in a sterile flask, and melt the agar by microwave. Add appropriate amounts of antibiotic and repour plates.