Replication and stability of the linear plasmid pBSSB2

Sunjukta Ahsan, Apollo-University Of Cambridge Repository, Apollo-University Of Cambridge Repository
2012
Plasmid pBSSB1 is a 27 kb linear DNA with proteins attached at the 5' termini. It encodes the H: z66 flagellar antigen in Salmonella enterica serovar Typhi (S. Typhi) isolated from Indonesia. Together with the H: j or H: d flagellar antigen encoded by the host chromosome, pBSSB1 renders expression of the flagellar antigen biphasic in S. Typhi. Following the discovery of pBSSB1, initial bioinformatic analyses were carried out. However, no genetic analysis of replication and stability functions
more » ... ability functions was conducted. Such studies form the basis of the present work. Plasmid pBSSB2, that contains a kanamycin cassette inserted at position 1295 bp of pBSSB1, was used in the present investigation. The first objective of the work was to develop a method of purification for the linear plasmid. Conventional plasmid extraction methods which had been used previously were found to produce a very poor yield of plasmid DNA. It was shown in the present study that a proteinase-K treatment was essential for the removal of the linear plasmid terminal proteins to avoid loss of the plasmid in the phenol-chloroform-isoamylalcohol treatment which removes cellular proteins from the plasmid DNA. The region containing the basic replicon of pBSSB2 was identified by screening for a region that was able to support replication in E. coli of a ColE1-like plasmid in a polA host (in which it would not normally replicate). This identified a 2831 bp fragment encompassing nucleotides 12820 to 15649 of pBSSB2. It was expected that this would encode an initiator of replication such as a Rep protein. However, mutagenesis studies showed that none of the annotated ORFs in this fragment was essential for replication. Candidate ORFs, not identified in the original annotation, have been suggested that remain to be tested as possible candidates for the rep encoding gene. The possibility of an alternative RNA primed initiation of replication has also been hypothesized. An adjacent region was found to exert strong incompatibility against pBSSB2, suggesting that it mi [...]
doi:10.17863/cam.16329 fatcat:g3wlm6hkz5c5bl62q5ezebsari