Methods for use with the mTn-3xHA/lacZ-mutagenized library

The following protocols are included:

• Making library DNA from the DNA we send you
• Transforming yeast with DNA from the insertion library
• Screening for gene expression using lacZ fusions
• Identification of the genomic site of transposon insertion
• Transferring the disruption allele to other strains
• Using the HAT epitope-tagging feature

DNA>Making library DNA from the DNA we send you

The original library was distributed as 18 individual pools. After 8/98, 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 -70^oC stock of this eluate. Dilute eluate into LB plus antibiotic to give a culture with an almost saturated density. Grow at 37^oC for a few hours. Make miniprep or midiprep DNA.

Transforming yeast with DNA from the insertion library

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 8/98, 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.

1. Plasmid DNA from pools of the mTn-3xHA/lacZ-mutagenized genomic library is digested with NotI. A 2.1-kb band from the vector should be very apparent, together with a broad band in the 8-kb region, representing inserts. Because sized genomic DNA was used to make the library, the insert bands are not very heterogeneous in size.
2. A 10-ml culture of the yeast host strain is grown to a density of 10⁷ cells/ml (O.D. 600 of 1). Use of such logarithmically-dividing cultures increases transformation efficiency.
3. Cells are pelleted and washed once with 5 volumes of One Step buffer (0.2M LiAc, 40% PEG 4000, 100 mM beta-mercaptoethanol). This wash is especially important when culture volumes are increased. Cells are resuspended in 1 ml of One Step buffer containing 1 mg of denatured salmon sperm DNA. 100 ul aliquots of this suspension are then added to tubes containing from 0.1 to 1 ug of NotI digested plasmid DNA.
4. Tubes are vortexed to mix the contents thoroughly, then incubated at 45^oC for 30 minutes.
5. Cells are pelleted and resuspended in 400 ul of SC-ura. 200 ul is plated onto SC-ura medium. Plates are incubated at 30^oC for 3 to 4 days.

Screening for gene expression using lacZ fusions

OVERVIEW: Transformant strains carrying in-frame fusions between yeast genes and lacZ are identified by a color assay for beta-galactosidase activity.

1. To maximize detection of lacZ fusions expressed at a low level, transformant colonies can be patched to YPAD plates at a density of 100 per plate.
2. To identify vegetatively expressed genes, cells are replica plated to an SC-ura plate on which a sterile disc of Whatman 1A filter paper has been placed, and grown overnight at 30^oC. Other media or growth conditions can be substituted as desired. For ade2 strains, any test media should contain 80 mg/l of adenine.
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-beta-D-galactopyranoside, 0.1 M NaPO4 [pH 7] and 1 mM MgSO4 in 1.6% agar) and incubated at 30^oC for up to 2 days. These plates can be very thin; their use increases the signal over that obtained by simply soaking the filters in a buffered X-gal solution.
5. Transformants carrying productive lacZ fusions are recovered from the regrown YPAD plates. It is advisable to subsequently maintain selection for URA3 wherever possible, as some mutations are deleterious even in the heterozygous state.

Identification of the genomic site of transposon insertion

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 rescue protocol for lacZ-based transposons. I have transcribed Carl's protocol and modified the suggested enzymes and primers, to make it suitable for PCR.html">vectorette PCR of the mTn-3xHA-based transposons.PCR

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.