mTn-lacZ/LEU2

TR	Tn3 terminal inverted repeats
lacZ	5'-truncated lacZ gene encoding beta-galactosidase
LEU2	LEU2 gene from S. cerevisiae
amp	Encodes beta-lactamase
loxP	lox site, target for Cre recombinase

Uses: Gene disruption, analysis of gene expression, immunodetection of beta-gal fusion protein.

In more detail: mTn-lacZ/leu2 was constructed by Siefert et al. (1986) . It 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 fusion protein can also be immunodetected using antibodies directed against beta-gal.

The accession for mTn-lacZ/LEU2 is U35112

A kit for mutagenesis of a yeast gene with mTn-lacZ/LEU2 is available.

• See what it contains
• Order the kit

Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-lacZ/LEU2

Please read this whole document before you start!

Shuttle mutagenesis

1. Clone fragment into vector pHSS6 (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-lacZ/LEU2 into cells by mating with strain B198
   • Grow strains overnight with antibiotic selection (Amp50 for B198).
   • Subculture 1:100 in fresh medium (no antibiotics). Grow at 37^o C 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^o C without agitation for 20 min to 1 hr. Plate as 100 ul aliquots onto LB Amp50 Kan40 Cm34.
   • Grow 1-2 days at 30^o C. Now have cointegrates. (Set up strain R1230 in Sm50 overnight).
4. Mate to strain R1230 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 R1230 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 Amp50 Kan40 Sm50 and grow overnight at 37^o C.
   • 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 Amp50 Kan40 to give an almost saturated density. Grow at 37^o C 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 endA cloning strain (eg DH5). Plate on LB Amp50 Kan40.
6. Transform into yeast selecting for LEU2
   • Elute entire pool of transformants (again, aim for thousands) and make a miniprep as in step 5. (Make -70 stock of bacterial pool for future use).
   • Transform Not I digest of entire pool into yeast and look for lacZ fusions among transformants.

Screening for in-frame lacZ fusions in yeast

1. Transformant colonies are patched to SC -leu.
2. Cells are replica plated to a SC -leu plate and a SC-leu plate on which a sterile disc of Whatman 1A filter paper has been placed, and grown overnight at 30^o C. 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.
3. 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.
4. 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 30^o C for up to 2 days.
5. Transformants carrying productive lacZ fusions are recovered from the regrown SC-leu plate . It is advisable to subsequently maintain selection for LEU2 wherever possible, as some mutations are deleterious even in the heterozygous state.

Bacterial strains used (included in kit):

R1123	Strain XL1-blue carrying vector pHSS6.
R1236/B211	Strain RDP146 (F- recA' (delta lac-pro) rpsE ; spectinomycin resistant) with plasmid pLB101 (pACYC184 with tnpA ; active transposase, chloramphenicol resistant)(F. Heffron)
B198	Strain RDP146 with pOX38 F factor derivative carrying mTn3 derivative mTn-lacZ/LEU2 (lacZ , LEU2, amp, ampicillin resistant)
R1230/NS2114Sm	F- recA rpsL (Streptomycin resistant, lambda-cre lysogen)

Vector used:

The accession for pHSS6 is M84115

Antibiotics used:

Ampicillin, Amp,	50 mg/ ml in water. Use at 50 ug/ml (Amp50)
Kanamycin, Kan,	10 mg/ ml in water. Use at 40 ug/ml (Kan40)
Chloramphenicol, Cm,	34 mg/ml in ethanol. Use at 34 ug/ml (Cm34)
Streptomycin, Sm,	10 mg/ml in water. Use at 50 ug/ml (Sm50)

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.