Transformation of Neisseria gonorrhoeae

Materials

Gonococcal strain which is Opa-postive /Pili positive

PPM medium (15g proteose pepton, 5g NaCl, 4g KH 2PO4 1g K2HPO4, 1g soluble starch, ad 1l H20 ph: 7.5) + vitamin mix

cloned and sequenced analysed gonococcal DNA which contains the spectinomycin resistance cassette and the Neisserial DNA uptake sequence (DUS) (plasmid DNA

Procedure

• Ngo strain (O+/P+) were grown overnight (37°C) on GC agar plates in a humid atmosphere with 5% CO 2
• Next day colonies were resuspended in 1ml PPM medium/Vitamin mix for 2 hours at 37°C with an agitation at 220 rpm
• The fresh suspension was measured at OD550 nm and afterwards diluted to OD550 nm of 0.1
• 1 ml of this gonococcal suspension is then incubated with 1 μg of the gonococcal plasmid DNA and incubated for 6 hours at 37°C with an agitation of 100 rpm
• The transformed bacteria are than seeded (undiluted, 1:10 and 1:100 diluted) on selective GC- agar plates containing spectinomycin and incubate for 2 to 3 days