Then, two PCR products were connected by overlapping PCR with primers XbaBTU15FW and BTU13RVXho. The overlapping PCR produced SalI, SmaI and SpeI sites between BTU1_5′ and BTU1_3′. The PCR product was cloned into the XbaI and XhoI sites of pBlueScript SK(+) vector (Stratagene)
to produce pBB. The neo5-MTT1-5′-2 segment was amplified from pMNMM3 by the PrimeStar HS DNA GF120918 ic50 Polymerase with primers neo5_FW_Sal and MTT1_MCS_RV p38 inhibitors clinical trials and cloned into the SalI and SpeI site of pBB. Then, an XhoI site was introduced between XbaI and NotI sites by site-directed mutagenesis with primers pBNMB_addXhoS and pBNMB_addXhoAS to produce pBNMB. neo5-MTT1-5′-2-HA-cre segment of pMNMM3-HA-cre1 was excised out by SalI and MluI and cloned selleck screening library into the SalI and MluI site of pBNMB to produce pBNMB-HA-cre1. The plasmid map and the DNA sequence of pBNMB-HA-cre1 can be found in the Additional file 1. The CU427 wild-type strain was transformed with the BTU1-5′-neo5-MTT1-5′-2-HA-cre1-BTU1-3′ construct which was digested out from pBNMB-HA-cre1 and the transformants were selected using 100 μg/mL paromomycin. The endogenous BTU1 loci were replaced with the construct by phenotypic assortment and selection using increasing concentrations of paromomycin. Six strains were selected for further studies. Induction of Cre-mediated loxP recombination For the experiment shown in Fig. 2A, exponentially growing B2086 or CRE556 cells were cultured
in 1× SPP medium with or without 1 μg/mL CdCl2 for 1.5 hr, or starved B2086 or CRE556 cells were cultured in 10 mM Tris (pH 7.5) with or without 50 ng/mL CdCl2 for 1.5 hr.
For the experiment shown in Fig. 2B, CRE556 and loxP-neo4-loxP-EGFP-TWI1 strains, both pre-starved over night in 10 mM Tris (pH 7.5) were mated and 50 ng/mL CdCl2 was added to the culture at 3.5 hr post-mixing (hpm). At the times indicated, cells were collected for immunofluorescence staining. For the experiment shown in Fig. 3B, starved CRE556 cells were pre-treated with 50 ng/mL CdCl2 for 1.5 hours and then mated with starved these loxP-neo4-loxP-EGFP-TWI1 cells in 10 mM Tris (pH 7.5) in the presence of 25 ng/mL CdCl2. At 2, 4, 6 and 8 hpm, genomic DNA was extracted for loxP excision analysis. For the experiment shown in Fig. 4B, starved CRE556 cells were pre-treated with 50 ng/mL CdCl2 in 10 mM Tris (pH 7.5) for 1.5 hr and then mated with pre-starved loxP-neo4-loxP-EGFP-TWI1 strains in the presence of 25 ng/mL CdCl2. At 2 hpm, single mating pairs were isolated into drops of 1× SPP medium. Cells were observed about every 2 hr until 6 hpm; then, cells were cloned into fresh drops of 1× SPP medium, in cases where pairs had separated. Cells were cultured for 2 days at 30°C and established clones were cultured in 1 mL 1× SPP medium for ~24 hr. The cells were then inoculated into 1× SPP medium containing 1 μg/mL CdCl2. Clones growing at normal speed in this medium were chosen as candidates for loxP-neo4-loxP-EGFP-TWI1 strain derived cells.