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.
- 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.
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.
- 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).
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.
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.
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/
- 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.