lacZY gene fusion cassettes with Kan^Rresistance

Abstract

We have constructed a series of lacZY gene fusion cassettes containing KanR to create active P-galactosidase fusions in anK desired reading frame. The cassettes were created by inserting the Tn5 Kan gene into the unique NruI site present in lackt (1) of the lacZY fusion plasmids described by

Membrane Topology of the NixA Nickel Transporter of Helicobacter pylori : Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III

“…E. coli DH5␣ [supE44 ⌬lacU169 (80 lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] and E. coli MC1061 [hsdR araD139 ⌬(araABC-leu) ⌬lacX74 galU galK rpsL thi] were used as recipients of recombinant plasmids (43). Plasmids pUEF204 [carrying the H. pylori nixA gene cloned in pBluescript II SK(ϩ) (Stratagene)], pLKC480, and pRT733 have been described previously (39,46,47). Strains were maintained on Luria-Bertani agar or sheep blood agar containing the appropriate antibiotics and were stored at Ϫ70°C in Luria broth supplemented with 15% (vol/vol) glycerol or MuellerHinton broth supplemented with 4% horse serum and 15% (vol/vol) glycerol.…”

“…A series of 21 3Ј truncates of nixA were PCR amplified as EcoRI-SalI fragments (primer sequences available upon request) and ligated into vector pLKC480 (47) to create in-frame LacZ fusions or into vector pBAF to create in-frame PhoA fusions. LacZ fusion constructs were transformed into E. coli MC1061 cells and selected on Luria agar containing ampicillin (100 g/ml), kanamycin (50 g/ml), and X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) (40 g/ml), PhoA fusions were transformed into E. coli DH5␣ cells and selected on Luria agar containing ampicillin (100 g/ml) and XP (5-bromo-4-chloro-3-indolylphosphate) (40 g/ml).…”

The Elastin-binding Protein of Staphylococcus aureus(EbpS) Is Expressed at the Cell Surface as an Integral Membrane Protein and Not as a Cell Wall-associated Protein

“…The forward primer was complementary to DNA located 5Ј to the trc promoter in pKK233-2 and contained an EcoRI site. When combined with reverse primers containing a 5Ј-SalI site that were complementary to different sequences within ebpS, it was possible to generate a set of ebpS-lacZ and ebpS-phoA fusions by cloning between EcoRI and SalI sites in the fusion expression vectors pBAF (phoA) (30) and pLKC480 (lacZ) (31).…”

Functional and Topological Analysis of the Burkholderia cenocepacia Priming Glucosyltransferase BceB, Involved in the Biosynthesis of the Cepacian Exopolysaccharide

“…4A). A series of 3Ј truncated sections of the bceB nucleotide coding sequence were PCR amplified as EcoRI-SalI fragments that were ligated into vector pLKC480 (19) to create in-frame LacZ fusions or into vector pBAF (9) to create in-frame PhoA fusions. E. coli DH5␣ cells transformed with the fusion constructs or the cloning vectors were grown in Lennox broth containing 1 mM IPTG and 150 mg/liter of ampicillin, harvested at the exponential phase, and permeabilized as described previously (9).…”