Objective
DEVELOPMENT OF STABLE CLONING VECTORS IN AN IMPORTANT INDUSTRIAL MICROORGANISM : BACILLUS SUBTILIS. SUCH DEVELOPMENT HAS BEEN HINDERED SO FAR BY SEGREGATIONAL AND STRUCTURAL INSTABILITY OF RECOMBINANT PLASMIDS. INVESTIGATION OF THE MECHANISM OF PLASMID INSTABILITY IS 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.
The mechanism whereby genetic material is rearranged in, or entirely lost from cells was investigated and based on this knowledge, new systems which allow stable maintenance of foreign genetic material were developed. 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.
Many plasmids, which are used as cloning vectors in Gram positive bacteria, replicate by a rolling circle type mechanism. This mode of replication was found to be error prone and leads to structural instability at high frequency. Other plasmids, which replicate by a different mechanism, resulted in structural instability at a much lower frequency, making them ideal candidates for cloning vectors. It was found that a decrease in the efficiency with which plasmid replication occurs leads to segregational instability of the plasmid. An alternative to cloning genes on plasmids is to insert them into the chromosome. It was found that genes could be stably maintained in the chromosome of B subtilis. It was also found that genes could be more stably maintained in some chromosomal locations than in others. The main conclusion drawn from these studies is that deoxyribonucleic acid (DNA) replication has a profound effect on the stability of genetic information.
DEVELOPMENT OF STABLE HIGH-LEVEL EXPRESSION VECTORS WHICH WOULD REPLICATE IN BACILLUS WITH A CONSTITUTIVELY LOW AND INDUCIBLE HIGH COPY NUMBER AND A STRONG CONTROLLABLE PROMOTER.
IN PARTICULAR :
1. THE REGION INVOLVED IN THE STABLE MAINTENANCE OF THE BACILLUS ENDOGENOUS PLASMID PBAA1 WILL BE IDENTIFIED.
2. THIS REGION WILL BE COMBINED WITH REPLICONS HAVING A CONSTITUTIVELY LOW BUT INDUCIBLY HIGH COPY NUMBER.
3. IN ORDER TO CONSTRUCT STABLE EXPRESSION VECTORS, THERMOINDUCIBLE PROMOTERS (EITHER HEAT SHOCK PROMOTERS OR PROMOTERS UNDER THE CONTROL OF TEMPERATIVE SENSITIVE REPRESSORS) WILL BE CLONED ONTO THE STABLE REPLICON.
Fields of science
Programme(s)
Topic(s)
Data not availableCall for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
DUBLIN 2
Ireland