Cryptic plasmid pBf1 from Butyrivibrio fibrisolvens AR10: its use as a replicon for recombinant plasmids
1992
Ware, C.E. | Bauchop, T. | Hudman, J.F. | Gregg, K.
A cryptic 2.85 kb plasmid (pBf1) was isolated from the rumen bacterium Butyrivibrio fibrisolvens strain AR10, mapped with restriction endonucleases, and cleavage sites suitable for attachment to Escherichia coli plasmids were identified. AR10 was not able to be cured of pBf1 by growth at 42 degrees C or in 0.25 micrograms ampicillin/ml, but growth in 50 micrograms acridine orange/ml for three culture passages produced cured colonies at a frequency of < 1%. Chimeric plasmids were constructed by combining pBf1 with the E. coli plasmid pUC18, in addition to the clindamycin resistance gene from Bacteroides fragilis plasmid pDP1 (pCW2 and pCW3), or the CAT gene from E. coli plasmid pKK232-8 (pCK1). For plasmid construction, pBf1 was cleaved at two alternative restriction sites to increase the likelihood that replication control sequences would remain functional in at least one of the plasmids. Electroporation of AR10 yielded transformant populations that clearly maintained the plasmids and that appeared to express the ampicillinase gene of pUC18, although transformants were not readily selectable with any of the three antibiotics. The suitability of pBf1 as a replicon on which to base the construction of shuttle vectors was demonstrated clearly, by persistence of plasmid pCW3 in the absence of selective pressure, and the addition of appropriate selection factors is expected to yield practical transformation vectors.
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