Tn 4351 Unit 5 Lab Report
Tn 4351 was originally isolated from bacteroides fragilis [30] . The transposon was successfully introduced into Cytophaga succinicans, Flavobacterium meningosepticum, Flexibacter canadiansis, Flexibacter strain SFI and Sporocytophaga myxococcoides by conjugation [25]. Tn 4351carries two antibiotic resistance gene. One of the codes for resistance to erythromycin and clindamycin which is expressed in bactroides but not in E.Coli. The other gene codes for resistance in tetracycline and is expressed in aerobically grpwn E. coli, but not in anaerobically grpwn E. coli or in bacteroides. The transposon of Tn4351 was originally detected in E. coli which carried an unstable chimeric plasmid, pSS-2. The mobilization of pSS-2 from onestrain of E. coli …show more content…
chinesis. A construct of R751::Tn4351 (the physical map of R751::Tn4351 and restriction sites are shown in fig. 7) was selected for introduction into F. chinesis to discover if the introduction and insertion of the vector R751 and the transposition of T4351 into the F. chinesis chromosome by a triparental mating occurred. One parent was E. coli GJ342 which carried a helper plasmid, the second parent was E. coli HB101 which contained R751::Tn4351 and the third parent was the F. chinesis target strain. 189 colonies were isolated on LB agar plates which in passage in fresh media were able to grow in 200µgml-1 erythromycin. The erythromycin resistance gene is carried by Tn4351. erythromycin resistance colonies were transfer to LB agar containing 200µgml-1 thrimethoprim. Non of the colonies could grow in this medium and no free vector (R751) was obtained in plasmid miniprep. This indicates that no replication of R751 occurred. Colony blot hybridization was done separately to discover if Tn4351 and/or R751 had inserted into the chromosome of F. chinesis. Duplicated blots were probed separately with radiolabled pVOHI (for the detection of the Tn4351) and R751 (for the detection of the transposon delivery vector). pVOHI is a derivated of pBR328 that carries Tn4351 but there is no common sequence with R751. Approximately half of the colonies selected from the first screen were positive in the second screenfor the detection of Tn4351 (Fig. 8.a). A few colonies were positive for the detection of R751 (Fig. 8.b). A few mutant defective in spreading were isolated (Fig. 8.c) and some auxotroph mutants were also
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