Yale Genome Analysis Center, Yale University http://ygac.med.yale.edu/mtn/reagent/avail_reagents/4xHA_lacZ_info_p.stm 
| 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!
Shuttle mutagenesis
- 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
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