The library is distributed as 18 individual pools in the form of DNA. You will be sent about a microgram of each. Transform a suitable amount into E. coli (use any kanamycin- and tetracycline-sensitive strain suitable for making plasmid preps). Select transformants with 40 ug/ml kanamycin and/or 3ug/ml tetracycline (allow at least an hour for expression following transformation). Obtain 50,000 colonies for each pool. Elute colonies from plates in LB; make a -70oC stock of this eluate. Dilute eluate into LB plus antibiotic to give a culture with an almost saturated density. Grow at 37oC for a few hours. Make miniprep or midiprep DNA.
OVERVIEW: Mutagenized DNA from the library is excised from the bacterial vector. It is then transformed into a ura3 strain of yeast. This procedure is outlined in this figure. The best strategy is to screen a few thousand transformants from each pool. Use of a circle-zero strain will prevent recovery of insertions in the 2-micron plasmid. Screening 30, 000 transformants should give you 95% coverage of the yeast genome.
To minimize double integrants, transformations should contain the lowest amount of DNA practicable. We therefore recommend that a pilot experiment be performed to determine transformation efficiency of the strain, and conditions then be scaled up as appropriate. The pilot protocol given below uses a modified version of the method of Chen et al. (1992).. You should use whatever transformation protocol works best in your hands.
We have not done assays of GFP activity in yeast.
See Niedenthal et al (1996) for their methods.
We tested mTn-3xHA/GFP by mutagenesis of BDF1, 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.
To determine the site of transposon insertion, genomic DNA imediately adjacent to the transposon sequences must be rescued. We have not yet constructed a rescue vector for mTn-3XHA/GFP. If demand for the library is high we will construct one. Otherwise, we will be happy to provide reagents and information to another laboratory who wishes to construct it. Inverse PCR on genomic DNA could be used to recover the site of insertion. Alternatively, Carl Friddle has developed a 'vectorette PCR' rescue protocol for lacZ-based transposons. I have transcribed Carl's protocol and modified the suggested enzymes and primers, to make it suitable for vectorette PCR of the mTn-3xHA-based transposons.
When transposon insertion has created an in-frame fusion to GFP in the gene of interest, the transposon can be excized 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 in the protein. 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).
N.B. When tagging essential genes, the original strain transformed should obviously be diploid. You can dissect the popped-out version to see if the tagged gene is functional. Only believe a tag is lethal if it is complemented by the wild-type gene, and if several popout events give the same phenotype.
TR in upper case. loxR in bold.
GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggccattgaa ggtagaagag aaaatttgta cttccaaaga aagaaggccg ctatcgcttc ggataactcc tgctatacga agttatgggc ggccgtttac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctccg gccgcCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCC
|Tetracycline, 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)|
|Ampicillin, Amp (Sigma A9518)||50 mg/ml in water. Use at 50 ug/ml (Amp50)|