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Development of a plasmid reporter system to identify group B meningococcal genes specifically expressed in vivo utilising Cre/loxP site-specific recombination.

Borde, Hema Ann. (2001) Development of a plasmid reporter system to identify group B meningococcal genes specifically expressed in vivo utilising Cre/loxP site-specific recombination. Doctoral thesis, University of Surrey (United Kingdom)..

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Attempts to produce an effective vaccine against group B meningococcal disease have been unsuccessful due to poor immunogenicity of the capsular polysaccharide, and high variability in the currently identified outer membrane proteins. There is therefore a need to find other vaccine candidates. It is clear from studies of bacteria such as Salmonella typhimurium, that successful pathogens have the ability to specifically express genes in response to an in vivo environment. Many of these in vivo-expressed gene products will contribute to virulence of the organism and may therefore be target antigens for development of new vaccines. An in vivo promoter-probe strategy was developed using the Cre/lox recombination system to identify meningococcal promoters that are specifically expressed in vivo. The strategy employs the cre gene, which encodes a site-specific recombinase that targets two 34-base pair directly repeated lox sites. A vector, pUS1604, was constructed which contains two lox sites flanking a transcriptional terminator that separates the aph (encoding kanamycin resistance) coding sequence from its promoter. A promoterless ere gene was placed on a low copy number vector to limit the number of Cre molecules within each host bacterial cell. Expression of Cre is detected by its ability to recombine the two lox sites and reconstitute a functional kanamycin resistance gene. The system was initially tested in E. coli by cloning a known regulated promoter from the araBAD operon that is induced by L-arabinose and repressed by D-glucose. However, the promoter failed to tightly control the regulation of cre gene expression, resulting in partial loxP recombination, which produced a mixed population of streptomycin and kanamycin resistant cells within each colony. Also, attempts to place the system into the meningococcus were unsuccessful. Efforts were made to tighten the regulation of the cre gene by placing the system into a different E. coli strain and test the system using a library of meningococcal genomic DNA fragments, as a compromise to show the experiments intended to isolate promoters. A vector, pUS1623, was constructed that would place the cre gene under the control of external promoters. A group B meningococccal genomic library was cloned upstream of the promoterless cre gene, with the inserted DNA fragments ranging from 0.1-1kb in size. The library was introduced into the E.coli XLl-Blue strain carrying pUS1604, and the Cre/lox recombination event has been shown to behave as expected, yielding a proportion of kanamycin-resistant clones. This indicates that promoter-like sequences are driving Cre, which is seen through the kanamycin-resistant phenotype of the clone. Conversely, clones that remain streptomycin-resistant do not have promoterlike sequences driving the expression of Cre. This streptomycin-resistant pool of clones was used to inoculate 50% serum/50% PBS liquid medium to identify group B strain meningococcal promoter sequences that drive Cre expression in this environment. Several sequences found to induce Cre expression from the initial screen, and during the serum experiment were sequenced. The sequence data was next analysed for homology against the sequences on the group B meningococcal genome sequence database ( to identify the function of the contig from which the isolated sequence is found within the meningococcal genome. Finally, the sequences were analysed for prokaryote -35 and -10 consensus regions and orientated to determine the direction of transcription relative to the cre gene and also to genomic open reading frames, to determine the likelihood of isolating actual promoters. Although the experiments carried out in E. coli do not give an accurate picture of the regulatory gene pathways in the meningococcus, the results give an insight into the suitability of the system as regards to its use in an IVET strategy.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors :
Borde, Hema Ann.
Date : 2001
Contributors :
Depositing User : EPrints Services
Date Deposited : 09 Nov 2017 12:16
Last Modified : 20 Jun 2018 11:12

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