mTn-3xHA/GFP

 

TR	Tn3 terminal inverted repeats
Xa	Factor Xa cleavage recognition site
loxR	lox site, target for Cre recombinase
GFP	gene encoding Green Fluorescent Protein mutant p11
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, creating fusion to GFP, HAT epitope-tagging protein at range of sites, creating conditional alleles.

 

In more detail: mTn-3xHA/GFP can be used easily to create a library of insertions, each at a different site 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 GFP can be used to monitor and quantify gene expression, via assays for fluorescence activity. Localization of the GFP fusion protein can be examined by fluorescence microscopy. 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 GFP 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. The HAT tag allows immunodetection of the protein. Insertion of the HAT tag also has the potential to create conditionally-defective forms of the protein.

 

The accession number for mTn-3xHA/GFP is U54830 .

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

• See what the kit contains
• Order the kit

 

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

 

Please read this whole document before you start!

 

Shuttle mutagenesis Clone target gene into vector pHSS6.

• pHSS6 is in 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 target plasmid into competent cells of R1236/B211.

• Select on LB Kan40 Cm34.

Transfer F::mTn-3xHA/lacZ into cells by mating with strain #111/B428.

• Grow strains overnight with antibiotic selection (Tet3 for #111/B428).
• Subculture 1:100 in fresh LB medium (no antibiotics). Grow at 37o C to early log phase (when cell swirls are visible). The recipient strain (R1236/B211) can be denser than the donor (#111/B428).
• Mix 200 ul of each strain. Incubate at 37o C 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 30o C. Now you have cointegrates.
• Set up strain #70/B425 in Sm50 Cm34 overnight.

Resolve cointegrates by mating to strain #70/B425.

• 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/B425 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 37o C
• 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 Tet3 Kan40 to give an almost saturated density. Grow at 37o 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 Tet3 Kan40

Transform into yeast.

• 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, selecting for URA3 .
• NB For HAT epitope-tagging, you may want to pre-transform your yeast strain with pB227/GAL-cre (selecting LEU2 )

Screening for in-frame GFP fusions in yeast

We have not done assays of GFP activity in yeast.

See Niedenthal et al (1996) for their methods.

 

Analyzing GFP fusion protein localization in yeast

We tested mTn-3xHA/GFP by mutagenesis of the BDF1 gene, which encodes a chromatin-associated protein. We grew individual bdf1::mTn-3xHA/GFP transformants to a density of 107 cells/ml in SC-ura. The last four hours of growth were at room temperature, to allow formation of the GFP chromophore. Then we examined cells directly using a Leitz microscopy with a system 13 filter (this may not be optimal). In 4 of 38 transformants, we saw green fluorescence of the nucleus. Fixation and spheroplasting of the cells improved the signal-to-noise ratio.

 

Using the exision feature to HAT-epitope tag a protein

A leu2 ura3 GAL+ yeast strain is required. When transposon insertion has created an in-frame fusion to GFP in the gene of interest, the transposon can be excized by Cre-mediated recombination to leave a 274 bp insertion (sequence given below) containing the 3xHA tag. With the 5 base pair duplication caused by transposon insertion, this gives an in-frame 93 amino acid insertion. The popout event is mediated by cre recombinase and requires induction of the GAL1-10 promoter on galactose. Our strains grow poorly on galactose but give 80 to 100% popouts.

The HA triple tag can be detected by mouse monoclonal antibodies 12CA5 (Boehringer) or MMS101R (BAbCo, Richmond, California). These antibody recognise cross-reacting yeast proteins of about 55kD or110kD, respectively, and can give a spotty background on immunofluorescence. Despite this drawback, the 3xHA tag has been used extensively and successfully in yeast. A rabbit polyclonal antisera is also available (101c500; BabCo) but this was less reactive in the one instance we tried. Protocols for yeast immunofluorescence can be found here , or in Methods in Enzymology 194 (1991).

 

Transform strain with plasmid pB227/GAL-cre, selecting on SC-leu. Inoculate transformants into 2mls SC-ura-leu with 2% raffinose as carbon source, and grow to saturation. Dilute 1/100 into SC-leu with 2% galactose as carbon source. As a control also dilute 1/100 into SC-leu with 2% glucose as carbon source. Grow for 2 days (some strains induce without growing). If grown, dilute 1/100. Otherwise, proceed with undiluted culture. Spot a 10ul drop onto an FOA plate and streak it for single colonies (non-quantitative approach!). Alternatively, plate dilutions onto SC media and replica to SC-ura to identify Ura- colonies. The induced cultures should give 100x more Ura- cells than the control. PCR primers designed using the sequence given below can be used to determine position of the tag. The IR elements and palindromic loxR region should be avoided.

N.B. When tagging essential genes, the original strain transformed should obviously be diploid. You can dissect the HAT-tagged version to see if the tagged gene is functional. To be rigorous, only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.

 

Sequence of HAT tag (3xHA):

TR in upper case. loxR in bold .

GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggccattgaa ggtagaagag aaaatttgta cttccaaaga aagaaggccg ctatcgcttc ggataactcc tgctatacga agttat gggc ggccgtttac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctccg gccgcCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCC

 

Bacterial strains used (provided in kit):

 

R1123	Strain XL1-blue carrying vector pHSS6.
R1236/B211	Strain RDP146 (F- recA' dlac-pro) rpsE ; spectinomycin resistant) with plasmid pLB101 (pACYC184 with tnpA ; active transposase, chloramphenicol resistant)(F. Heffron)
#111/B428	Strain RDP146 with pOX38 F factor derivative carrying mTn3 derivative mTn-3xHA/GFP (GFP, URA3, tet ; tetracycline resistant)
#70/B425	Strain NG135 (K12 recA56 gal-delS165 strA ; streptomycin resistant) with plasmid pNG54 (pACYC184 with mTn3 res and tnpR seqs; active resolvase, chloramphenicol resistant)(N. Grindley)
B227	Strain DH5-alpha carrying pB227/GAL-cre (amp, ori, CEN, LEU2 ) (B. Sauer)

Vector used:

The accession for pHSS6 is M84115

 

Antibiotics used:

Tetracycline HCl, Tet (Sigma T3383)	12 mg/ ml in 50% ethanol. Use at 3 ug/ml (Tet3)
Kanamycin, Kan (Sigma K800)	10 mg/ ml in water. Use at 40 ug/ml (Kan40)
Chloramphenicol, Cm (Sigma C0378, I think)