|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.
Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-lacZ/LEU2
Please read this whole document before you start!
- 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.
- Transform this plasmid into competent cells of R1236/B211.
- 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 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 Amp50 Kan40 Cm34.
- Grow 1-2 days at 30oC. Now have cointegrates. (Set up strain R1230 in Sm50 overnight).
- 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 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 Amp50 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 endA cloning strain (eg DH5). Plate on LB Amp50 Kan40.
- 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 NotI digest of entire pool into yeast and look for lacZ fusions among transformants.
Screening for in-frame lacZ fusions in yeast
- Transformant colonies are patched to SC -leu.
- 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 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-leu plate . It is advisable to subsequently maintain selection for LEU2 wherever possible, as some mutations are deleterious even in the heterozygous state.
|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) |
The accession for pHSS6 is M84115
|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.