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mTn-3xHA/lacZ info/protocol


Diagram of 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.

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).
  2. Transform this plasmid into competent cells of R1236/B211.
  3. Transfer F::mTn-lacZ/LEU2 into cells by mating with strain B198
  4. Mate to strain R1230 to resolve cointegrates
  5. Rescue resolved DNA from this strain
  6. Transform into yeast selecting for LEU2

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 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.
  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 30oC 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.

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