Nigel Peter Minton

Improved plasmid vectors for the isolation of translational lac gene fusions

The beta-galactosidase fusion vector pMC1403 has been modified to include the unique cloning sites EcoRI, SmaI, BamHI, SalI, AccI, PstI and HindIII. The new vectors (pNM480, pNM481 and pNM482) allow the fusion of genes to beta-galactosidase in all three translational reading frames, and exhibit an increased sensitivity of promoter detection due to a higher copy number.

The complete amino acid sequence of the Clostridium botulinum type A neurotoxin, deduced by nucleotide sequence analysis of the encoding gene

A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the N-terminus of the Clostridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII-TaqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb ScaI-TaqI fragment and a 3.4-kb HpaI-PvuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149 502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.

Vectors for Use in Clostridium acetobutylicum

In the last two decades, interest in using surplus/waste biomass in biocatalytic processes to produce chemicals and fuels of high added value, as an alternative to chemical synthesis from petroleum feedstocks, has exhibited considerable fluctuation. The underlying argument for such developments seems irrefutable. Fossil fuels are a finite resource. It follows that alternative technologies based on a renewable feedstock must eventually be developed. Impetus for undertaking the necessary research has until relatively recently been ruled by economic factors. Thus, although the world oil crisis of the 1970s saw a surge in the fortunes of potential biological processes, a subsequent reduction in petroleum prices dampened initial enthusiasm. Two factors have now refocused the world’s attention on biocatalytic processes.