DEVELOPMENT OF STABLE CLONING VECTORS IN AN IMPORTANT INDUSTRIAL MICROORGANISM : BACILLUS SUBTILIS. SUCH DEVELOPMENT HAS BEEN HINDERED SO FAR BY SEGREGATIONAL AND STRUCTURAL INSTABILITY.
THE INVESTIGATION OF THE MECHANISMS OF PLASMID INSTABILITY ARE REQUIRED FOR SUCCESSFULL GENETIC ENGINEERING NOT ONLY IN BACILLUS BUT ALSO IN OTHER POTENTIAL INDUSTRIAL GRAM POSITIVE BACTERIA.
The objectives of this project were to investigate the mechanism whereby genetic material is rearranged in, or entirely lost from cells, and based on this knowledge, to develop new systems which allow stable maintenance of foreign genetic material. The host organisms used were Escherichia coli and Bacillus subtilis. Particular emphasis was placed on investigation of the mode of replication of plasmids used as cloning vectors. The relationship between the efficiency of plasmid replication and segregational stability was examined. The fidelity with which certain sequences are duplicated when placed on plasmids which replicate by different mechanisms was evaluated. The stability of foreign genes inserted into different chromosomal locations was also studied.
P. aureofaciens SBW25, a plasmid free, nonpathogen, was the dominant species (<30%) of bacteria found in the phylloplane of sugar beet. SBW25 was chromosomally marked with pMOB::TN5XYLE, to confirm the suitability of SWB25, the markers (Kmr, XylE) and to develop the plant model. Recombinants' free of any mobilisable elements or plasmid sequence were made; SBW25 marked by the insertion of a Kmr gene (From Tn903) into a known, nonfunctional chromosomal site to create safe, environmentally acceptable genetically modified organisms (GMOs) as competitively fit as the wild type. Recombinants were indistinguishable from wild type in mono or mixed cultures, with no loss of phenotype recorded over 500 generations in continuous culture of 250 days In planta. SBW25 marked at the Ee site with an integration vector (pBGS)(SBW25EeKmXylE) was equally stable.
A reliable method of transforming B. thuringiensis was developed. The segregational stability of the markers XylE and Gus, positioned on plasmid molecules under the control of Spo2 or PRLB promoters, was assessed in B. thuringiensis, Gus was unstable and therefore unsuitable, XylE and Mer were the preferred non-antibiotic markers.
STUDY OF THE MECHANISMS FOR PLASMIDIAL STRUCTURAL INSTABILITY IN BACILLUS SUBTILIS.
IN PARTICULAR :
1- THE RECOMBINATION MECHANISM STIMULATED BY PLASMIDS SINGLE STRANDED DNA SYNTHESIS WILL BE ASSESSED.
2- THE RECOMBINATION MECHANISM STIMULATED BY INTERRUPTIONS OF A DNA STRAND WILL BE STUDIED.
3- PLASMIDS WHICH REPLICATE WITHOUT STIMULATING RECOMBINATIONS WILL BE USED TO CONSTRUCT CLONING VECTORS BY CONVENTIONAL METHODS.
Funding SchemeCSC - Cost-sharing contracts