The experiments performed on B. fragilis used a custom transposome generated using the pMOD-3 [R6Kγ/MCS] vector. Screening/antibiotic resistance markers for erythromycin and kanamycin were introduced into the vector. As Bacteroides has a restriction system that requires bypassing, the pMOD3 construct (referred to as pVY02) was passaged through a strain of B. fragilis to methylate and protect the plasmid containing the transposon construct in a process called "transposon laundering" (Barry Hall, University of Rochester, personal communication). The transposon was released and purified from pYV02 using restriction digestion with Pvu II, gel-purified, and used to prepare the transposome complex with EZ-Tn5 Transposase. The transposome was electroporated into B. fragilis strain BF638 using standard methods and transposition mutants were recovered by screening on Brain/Heart Infusion (BHI) agar/erythromycin plates.
Results showed the transposon inserted in vivo at a relatively high frequency into BF638R, yielding 3.2 ± 0.35 x 10^3 CFU/µg. Transposome laundering resulted in a 6-fold increase in the number of transposome mutants over the nonprotected transposome controls. The use of the EZ-Tn5 pMOD-3 vector in the transposome construction process facilitated marker rescue by restriction digestion of DNA from the transposed host, recircularization, and transformation of the rescued DNA in E. coli strain EC100D (pir+). The transposome inserted into the Bacteroides genome in a single copy, demonstrated by Southern blotting and by single random-primer PCR (similar to the Random Amplification of Transposome Ends method). Further work by the researchers also demonstrated that the custom transposome was also useful in the mutagenesis of the bacterium Bacteroides thetaiotaomicron.
Veeranagouda, Y et al. (2012). Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiology Letters PMID: 22639975