Tn5 mutagenesis and mapping A library of transposons

Tn5 mutagenesis and mapping A library of transposons selleck kinase inhibitor in YS1646 was made using the EZ::TN insertion kit from Epicentre (Madison, WI). Over 56,000 kanamycin resistant (KanR) clones of YS1646 were pooled. The pool was screened for mutation rate

and auxotrophy for different biosynthetic pathways by replica plating onto minimal media and media containing various pools of amino acids and bases [30]. Following selection for CO2 resistance by plating dilutions to LB-Kan and incubating in 5% CO2, the colonies were again pooled and a P22 lysate was generated and transduced to a non-suppressed strain and purified for kanamycin resistance under non-CO2 conditions in order to separate spontaneous mutants from Tn5-based suppressors. Transposon-associated Tn5 insertions were identified by replica plating in air and CO2. Mapping of the insertion sites was performed by using the GenomeWalker™ kit (Clonetech, Mountain View, CA) according

to the manufacture’s instructions. Construction of non-polar deletion in zwf A non-polar deletion in zwf was generated by constructing a pCVD442 vector capable of deleting the entire zwf coding region by homologous recombination with the Salmonella chromosome [10]. selleck screening library Primers for PCR were designed that would generate one product immediately upstream of the 5′ ATG start codon and a separate product immediately downstream of the 3′ stop codon of the zwf coding region. The two separate products could then be ligated sequentially into the pCVD442 vector. The primers were: zwf-5′-reverse: 5′-GTGTGAGCTCGTGGCTTCGCGCGCCAGCGG

CGTTCCAGC-3′ (with added SacI), zwf-5′-forward: 5′-GTGTGCATGCGGGGGG CCATATAGGCCGGGGATTTAAATGTCATTCTCCTTAGTTAATCTCCTGG-3′ (with added SphI), zwf-3′ reverse: 5′-GTGTGCATGCGGGGTTAATTAA GGGGGCGGCCGCATTTGCCACTCACTCTTAGGTGG-3′, and zwf-3′-forward: 5′-GTGTGTCGACCCTCGCGCAGCGGCGCATCCGGATGC-3′). The primers also oxyclozanide generate internal NotI, PacI, SphI, SfiI, and SwaI in order to facilitate cloning of DNA fragments into the Δzwf for stable chromosomal integration without antibiotic resistance. This vector is referred to as pCVD442-Δzwf. The presence of the deletion, in AmpS SucR colonies, was detected by PCR using the following primers:zwf-FL-forward: 5′-ATATTACTCCTGGCGACTGC-3′ and zwf-FL-reverse: Selleckchem 10058-F4 5′-CGACAATACGCTGTGTTACG-3′. Wild type produces a 2,026 base pair product whereas the mutant produces a 608 base pair (bp) product, a difference of 1418 bp, which corresponds to the size of the zwf gene (1475 bp minus a 57 bp multiple cloning site that replaces the open reading frame). β-galactosidase Assay For β-galactosidase expression, lacZ was cloned into the high copy vector pSP72 (Promega) in E. coli, transformed into Salmonella strains (via restriction defective Salmonella strain YS501 [31], and screened for bright blue colonies on LB agar containing 40 μg/ml X-gal. lacZ was cloned from E. coli K-12 MG1655 [32] obtained from the Yale E.

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