pBendBlue: modification of the pBend system for color selectability.

Abstract

The binding of proteins to specific DNA sequences frequently results in localized bending of the DNA, which may play important roles in regulating transcription of a variety of genes (1,3). The pBend family of plasmids (2,4) allow generation of permuted fragments containing cloned DNA-binding sites and provide a convenient and widely used method for analyzing DNA bending. In most popular cloning vectors, blue/white color selection based on expression of the Escherichia coli lacZ gene is used to facilitate identification of clones containing DNA inserts. We report that color selection can also be extended to the pBend system. We observed that the permutation element (PE) of pBend2 (2) contains a single open reading frame (ORF) in one direction, starting with the second base of the EcoRI site (Figure 1) and another on the complementary strand, starting with the second base of the HindIII site (not shown). This suggested to us that the PE could be cloned into a color-selectable vector, such as pBluescript (Stratagene, La Jolla, CA, USA) and put in-frame with the lacZ color-selectable marker gene. The lacZ ORF in the pBluescript II SK(+/-) phagemid begins with methionine at base pair 816. Several of the restriction sites within the pBend2 PE do not occur in pBluescript (i.e., BglII, MluI, NcoI, NheI, NruI, StuI and StyI). Of the sites occurring in both, the PE fragments have clearly different sizes compared with those from pBluescript (DraI, PvuII, RsaI and SspI). Remaining common sites (BamHI, ClaI, EcoRI, EcoRV, HindIII, SmaI, SpeI, XhoI, XbaI and SalI) were eliminated by digestion of the pBluescript vector at the SacI (SstI) and ApaI sites, removing bases 663–759 of the multiple cloning site. The PE was then inserted into the SstI/ApaI sites. The 241-bp PE of pBend2 was polymerase chain reaction (PCR)-amplified using primers containing the SstI and ApaI restriction sites and additional bases to maintain an ORF upon ligation into pBluescript. The 5′ primer (5′tcgtcttcaaGAGCTCGAATTCACGCGTAG-3′) contained 10 nucleotides (nt) of pBend2 5′ to the EcoRI site (lower case), an SstI site (underlined) and the first 14 bases of the 241-bp PE (bold). The 3′ oligonucleotide (5′ctaccgcattaGGGCCCGAAGCTTGGATCC-3′) contained 11 nt of the original pBend2 3′ to the HindIII site (lower case), an ApaI site (underlined), a single G to maintain the ORF (italics) and the last 12 nt of the PE itself (bold). For improved PCR, pBend2 was chemically denatured prior to PCR. One microgram of plasmid DNA was resuspended in 18 μL of H2O, then denatured by incubation with 2 μL 2 N NaOH for 5 min at room temperature. The reaction was terminated by addition of 3 μL 3 M sodium acetate (pH 5.0), 7 μL H2O and 75 μL ethanol followed by 15 min on dry ice and pelleted by microcentrifugation for 15 min. The DNA pellet was washed with 1 mL 70% (vol/vol) ethanol, pelleted and dried. The pellet was resuspended in water and used for PCR in the following reaction mixture: 100 ng denatured template DNA, 5 μL 10 mM 5′ primer, 5 μL 10 mM 3′ primer, 8 μL of 2.5 mM of a dNTP mixture, 10 μL Expand High Fidelity 10× reaction buffer with 15 mM MgCl2 and 1 μL Expand High Fidelity Taq DNA Polymerase (Boehringer Mannheim, Indianapolis, IN, USA) in a 100-μL reaction volume. Amplification used incubation at 94°C for 1 min followed by 30 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min. This protocol produced a single PCR product that was cloned using the pCR 2.1 System (Invitrogen, Carlsbad, CA, USA) and sequenced. The modified PE was digested with ApaI and SstI. pBluescript was also cut with ApaI and SstI and dephosphorylated with calf intestinal alkaline phosphatase (CIAP) following the manufacturer’s protocol (Life Technologies, Gaithersburg, MD, USA). The digested PE fragment and pBluescript plasmid were ligated using a 3:1 molar ratio of insert to plasmid for 1.5 h at 16°C using the Takara DNA Ligation Kit (PanVera, Madison, WI, USA). Following the manufacturer’s instructions, 20 μL of the ligation mixture were used to transform 100 μL of Epicurian Coli SURE2 supercompetent cells (Stratagene) (used to reduce rearrangements or deletions resulting from repeated sequences within the PE). Cells were plated onto LB agar plates containing 50 μg/μL ampicillin and coated with

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Cite this paper

@article{Sperbeck1998pBendBlueMO, title={pBendBlue: modification of the pBend system for color selectability.}, author={S J Sperbeck and Graeme J. Wistow}, journal={BioTechniques}, year={1998}, volume={24 1}, pages={66-8} }