The original library was distributed as 18 individual pools. After , there is a new library that is distributed as 10 pools. You will be sent about a microgram of each pool in the form of DNA. 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. For libraries distributed after , the genomic library was made from a circle-zero strain (also rho-minus) so this is not necessary. 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.
OVERVIEW: Transformant strains carrying in-frame fusions between yeast genes and lacZ are identified by a color assay for beta-galactosidase activity.
OVERVIEW: To determine the site of transposon insertion, genomic DNA imediately adjacent to the lacZ sequences is rescued in Escherischia coli (see figure). To introduce an origin of replication (ori), a plasmid marked with LEU2 (pRSQ2-LEU2, U64693) replaces part of the transposon by recombination between plasmid- and transposon-borne copies of lacZ sequences. Yeast DNA is recovered from these transformants and cut with a 'recovery' enzyme (EcoRI, HindIII, ClaI, SalI, XhoI, KpnI). This releases as a linear segment the bacterial origin of replication, the beta-lactamase gene and a portion of the lacZ gene with adjacent yeast DNA; this fragment is then circularized and recovered in bacteria. pRSQ2 is high copy number in E. coli. Plasmids are sequenced using a primer complementary to the 5' end of the transposon. This process requires the three protocols given below.
Alternative method: Carl Friddle (http://genome-www.stanford.edu/group/botlab) 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.
CAUTION! Two or more insertion events may have occurred. These can be identified by examination of segregation of the transposon-borne URA3 marker upon tetrad dissection. You should be sure that the strain has only one transposon before proceeding to recovering a plasmid containing genomic DNA. If you have used the library for mutagenesis, you are strongly advised to make sure that your phenotype is linked to the transposon insertion, since spontaneous mutations can arise at other sites. You can waste time recovering junk if you don't check.
We suggest transforming the yeast with 1-5ug of BamHI-digested pRSQ2-LEU plasmid DNA, selecting the transformants on SC-leu-ura. This is a targetted replacement, so efficiency will depend on your strain. The method of Chen et al. (1992) given above can be used. If an ampR plasmid is present in the yeast strain to be transformed, a different marker could be cloned into the pRSQ2 polylinker to enable its recovery.
For a detailed discussion of genomic DNA preparation from yeast, see Philippsen et al. (1991). Here is the method that we use.
Upper case indicates terminal repeat element also present in vector:
The accession for mTn-3xHA/lacZ is U54828.
When pRSQ2-LEU2 integrates into mTn-3xHA it creates an 11.8 kb insertion. This element is not cleaved by the following enzymes: AscI, AvrII, BspEI, MscI, NotI, PmeI, PmlI, SacII, SnaBI, SpeI. These enzymes can therefore be used to recover a large plasmid containing sequences both 5' and 3' to the transposon insertion. We have successfully 'moved' disruptions by this strategy.
When transposon insertion has created an in-frame fusion to lacZ 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 or 110kD, 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)|