Patented Technologies

Synthetic Operon Construction in Clostridia Allele Coupled Exchange (ACE)

WO 2009/101400

An homologous recombination system that avoids the need for a heterologous reporter system and which enables large tracts of DNA to be inserted into a host bacterium such as Clostridium acetylbutylicum.

Academic Publication

  • Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. John T. Heap, Muhammad Ehsaan, Clare M. Cooksley, Yen-Kuan Ng, Stephen T. Cartman, Klaus Winzer, and Nigel P. Minton* Nucleic Acids Res. (2012); 40(8): e59.
  • Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles. Ng YK, Ehsaan M, Philip S, Collery MM, Janoir C, Collignon A, Cartman ST and Minton NP, (2013). PloS one. 8(2), e56051.

A Negative/Counter Selection Marker for Use in Clostridia: Cod A Selectable Marker

WO 2010/084349

The cytosine deaminase gene (codA) of Escherichia coli as a heterologous counter-selection marker for genetic manipulation of wild-type C. difficile strains. CodA not only converts cytosine to uracil but also converts the innocuous pyrimidine analog 5-fluorocytosine (FC) into the highly toxic 5-fluorouracil (FU) leading to incorporation of fluorinated nucleotides into DNA and RNA. It is this latter activity which allows CodA to be an effective counter-selection marker.

Academic Publication

  • Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between tcdC genotype and toxin production. Applied Environmental Microbiology 78: 4683–90 Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP (2012).

Transposon Delivery System for Clostridia (vector) TraDis Transposon System

WO 2013/144653

A plasmid-based transposon system in which Clostridial cells transformed with the plasmid results in cut and paste integration of the transposon into chromosomal DNA with a selectable marker. Loss of the plasmid in non-permissive conditions (using inducible promoters) prevents further transposition and facilitates selection of transposon-carrying colonies and the creation of transposon libraries. The patent facilitates TraDIS (Langridge et al. (2009) Genome Res. 19: 2308-2316.

Orthogonal Bacterial Expression System

WO 2013/144647

An expression system exploiting Group 5 RNA polymerase sigma factors for example the TcdR Sigma factor from Clostridium difficile. Such factors are poorly recognised in bacteria lacking Group 5 RNA Polymerase. An example of an orthogonal construct would be TcdR operably linked to a promoter recognised by TcdR and a heterologous DNA sequence in a bacterial host lacking any Group 5 RNA Polymerase and as such give constitutive expression of any TcdR-promoter linked DNA sequence.

ClosTron

US 20110124109 (US-only patent)

A type II intron system based on TargeTron which enables gene knock-outs through intron insertion in Clostridia. Visit ClosTron at http://clostron.com/

Academic Publication

  • The ClosTron: A universal gene knock-out system for the genus Clostridium. Heap, J.T., Pennington, O.J., Cartman, S.T., Carter, G.P. and Minton, N.P., 2007. Journal of Microbiological Methods. 70(3), 452-464.
  • The ClosTron: Mutagenesis in Clostridium refined and streamlined. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. J Microbiol Methods. 2010 Jan;80(1):49-55.