A negative / counter selection marker for use in clostridia


Genetic manipulation of Clostridium


A research team led by Prof Nigel Minton has developed ways to engineer species of Clostridium to either introduce news genes into their chromosome or to delete gene sequences from their genome. The Clostridia have until now been recalcitrant species for gene manipulation, however, our new technology enables Clostridia to be manipulated for industrial, research and other applied uses. We have three current patent applications to protect various aspects of the technology. By way of example one is outlined here.
This technology enables the sequential insertion of genes into the bacterial genome with positive selection at each stage to enable the construction of e.g. artificial operons.

Key Benefits

Hitherto it has not been possible to manipulate the genomes of Clostridia in a targeted and controlled way. Using Prof Minton’s technologies it is now possible to introduce new enzyme encoding genes into species of Clostridia, to use homologous recombination to modify existing gene sequences and in general be able to modify the genomic sequence of Clostridia in much the same way as E.coli and other bacterial species have been routinely modified.

IP Status

This patent is currently in PCT published as WO2009/101400. The University is looking to licence this technology and two related patented technologies for the genetic manipulation of Clostridia in a range of fields.

Technical Information

Recombination may occur at any of the homology arms (A), (B) and (C) of the plasmid and the corresponding homology arms of the chromosome.  Here the cargo DNA is represented as Bio-Bricks 1-4.  The cargo sequence may include an appropriate promoter to direct transcription of the pyrZ and pyrD ORFs in the desired final recombinant cell, as illustrated. The phenotype of the host cell is indicated in box.  The phenotype that the plasmid confers on the host cell is also indicated in a box.  In step 1 indicated by the first arrow, a first homologous recombination event occurs between the plasmid and the chromosome.  Possible products of the first recombination event resulting from homologous recombination at either one of the three pairs of homology arms (A), (B) or (C) are illustrated, together with the phenotype conferred on the host cell.  For each first recombination product, pairs of homology arms which are able to mediate a second recombination event are indicated by thin dashed lines.  Adjacent to each dotted line is a circle containing a “+” or a “–“ sign.  The “+” sign indicates that the product of homologous recombination at the relevant homology arms confers uracil prototrophy.  The “-“ sign indicates that the host cell containing the relevant product is a uracil auxotroph.  The desired product of the second recombination event can be obtained from the type (C) first recombination product, by homologous recombination at the pair of homology arms indicated with thick dashed lines.  In the second step indicated by the arrow, this desired product is formed as illustrated.  The phenotype of the cell containing this product is indicated in the box.  Key: “uracil –ve” means uracil auxotroph; “uracil +ve” means uracil prototroph; “Tm” means thiamphenicol; “FOA” means fluoroorotic acid; “R” means resistance phenotype; “S” means sensitive phenotype; “ori” is the origin of replication of the plasmid.

Patent Information:
For Information, Contact:
Jonathan Gibbons
Senior Licensing Executive - Healthcare
The University of Nottingham
+44 (0) 115 82 32189
Stephen Cartman
Nigel Minton
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