Hybond n plus nylon membrane

Tn5 mutagenesis was performed by using triparental matings between donor E. coli (pSUP2021) containing the transposon Tn5 (Km resistance), a helper E. coli strain (pRK2013) and recipient Pfv UPB0736 strain. Briefly, Pfv UPB0736, donor and helper E. coli strains were grown overnight in 20 ml of LB media supplemented with appropriate antibiotics. Cells were pelleted, washed and re-suspended in 10 ml of sterile LB media. Absorbance of all three strains were measured and cells were mixed in the following ratio: recipient Pfv UPB0736, 2×10⁸ colony forming units (cfu/ml); helper E. coli, 4×10⁹ cfu/ml; donor E. coli, 4×10⁹ cfu/ml. The mixture of cells were pelleted out, re-suspended in small volume of LB media and spotted onto Hybond N Plus nylon membrane (Amersham Pharmacia Biotech) that was overlaid on LB agar. Overnight incubated cells grown at 28°C were scraped from the membrane and re-suspended in 1 mL of sterile LB media. The cell suspension (50 μl each) was plated on LB agar plates containing Nf and Km. The plates were incubated at 28°C for 2-3 days to allow the growth of transconjugants (Tn5 mutants). The Pfv Tn5 mutants were then patched onto LB agar plates with Nf and Km, grown in liquid media and a glycerol stock was prepared and stored at -80°C.

The transposon, Tn5gusA11 having a spectinomycin resistance marker was maintained in E. coli strain TP003 as a suicidal plasmid (36 (link)). Biparental mating between the donor E. coli TP003 and the recipient B. gladioli NGJ1 strain was performed, as described before (37 (link)). Briefly, a single colony of E. coli strain TP003 was mixed with the recipient B. gladioli NGJ1 cells on a piece of Hybond N Plus nylon membrane (Amersham Life Science, Buckinghamshire, UK). Upon incubation for 48 h at 28°C, the bacterial cells were scraped with a sterile toothpick, resuspended in 100 μL sterile water and plated on KBA (King’s medium B Base; HiMedia, India) supplemented with rifampicin (50 μg/mL) and spectinomycin (50 μg/mL). The plates were incubated at 28°C and positive conjugants were analyzed for gus (β-glucuronidase) activity, as described in (36 (link)). Briefly, bacteria were taken in a sterile microcentrifuge tube, mixed with 1 μL of X-gluc (5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid;100 mg/mL) and incubated at 37°C for 30 min or until the appearance of blue color in the suspension.