Mutagenizing a yeast gene with mTn-3xHA/lacZ 【Yale University】

Yale Genome Analysis Center, Yale University

http://ygac.med.yale.edu/mtn/reagent/avail_reagents/3xHA_lacZ_info_p.stm

TR	Tn3 terminal inverted repeats
Xa	Factor Xa cleavage recognition site
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-3xHA/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 274-bp insertion containing sequences encoding the 3xHA tag and the factor Xa protease cleavage recognition site. When lacZ is fused in-frame to the gene of interest, the excision event results in an in-frame insertion of 93 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.

GenBank Accession: U54828.

A kit for mutagenesis of a yeast gene with mTn-3xHA/lacZ is available.

• See what it contains
• Order the kit

Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-3xHA/lacZ

Please read this whole document before you start

Shuttle mutagenesis

1. 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.

2. Transform this plasmid into competent cells of R1236/B211.

   • Select on LB Kan40 Cm34.
3. Transfer F::mTn-3xHA/lacZ into cells by mating with strain #95.
   • Grow strains overnight with antibiotic selection (Tet3 for #95).
   • Subculture 1:100 in fresh medium (no antibiotics). Grow at 37^oC 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 37^oC 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 30^oC. Now you have cointegrates.
   • Set up strain #70 in Sm50 Cm34 overnight.
4. 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 37^oC.
   • Do the Control: Spot the starting strains onto this media.
5. 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 37
     

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