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Development of gene reporters to aid in the analysis of the physiological factors which affect botulinum toxin product - FS1529

in food
1. To develop and optimise gene transfer in a representative toxingenic strain of a Group II nonproteolytic C. botulinum and a non-toxinogenic variant of authentic genetic pedigree. 2. To construct gene fusions between the promoter regions of appropriate toxin genes and a promoterless copy of the Clostridium thermosulfurogenes lacZ gene. 3. To localise the constructed botulinum promoter: : b-galactosidase fusions genes to low copy clostridial shuttle vectors, introduce them into the non-toxinogneic variant and monitor enzyme activity under different growth conditions. 4. To develop integrative procedures for each toxinogenic type, and demonstrate gene replacement by double cross-over. 5. To adapt the promoter: : lacZ gene fusions constructed under (2) with the integrative procedures developed under (4) to bring about the chromosomal replacement of the toxin stuctural gene with lacZ. 6. To use the lacZ integrants to study b-galactosidase production in the resultant integrants under various physiological conditions. 7. To construct an artifical operon, under the transcriptional control of a single promoter, composed of the two genes encoding acetoacetate decarboxylase and acetoacetyl-CoA: acetate butyrate:CoA transperase. Once constucted, introduce the operon on a suitable shuttle vector into representative Gp I C. botulinum strain, and establish whether acetone is produced. 8. To place the constructed "acetone" operon under the control of a toxin gene promoter and demonstrate that acetone production is stimulated/depressed by the same conditions shown to effect b-galactosidase production shown in (3) & (5).
Time-Scale and Cost
From: 1996

To: 1999

Cost: £221,124
Contractor / Funded Organisations
Centre for Applied Microbiology and Research