Clostridioides difficile
A series of pyrE-basedACE vectors are available equivalent to those made for C. acetobutylicum. All carry the C. perfringens catP gene conferring thiamphenicol resistance; the replication region of the E.coli plasmid ColE1, and the TraJ transfer function of the RP4 oriT region. Plasmid pMTL-YN1, and derivatives, carry the pCB102 Gram +ve replicon and is intended for use in the 630Δerm strain, whereas plasmid pMTL-YN2, and derivatives, carries the pBP1 Gm +ve replicon and is intended for use in RT027 strains, such as R20291 [1].
Correction Vectors pMTL-YN1 and pMTLYN2
Used to restore the mutant pyrE locus to wildtype – intended for use when the phenotype of mutations made elsewhere in the genome, using the KO vectors pMTL-YN3 and pMTL-YN4, needs to be assessed. Equivalent to the C. acetobutylicum plasmid pMTL-ME6 [2].
Complementation Vectors pMTL-YN1C and pMTLYN2C
Used to deliver a functional copy of a knocked-out gene (using pMTL-YN3 and pMTL-YN4) into the genome at the pyrE locus, while at the same time restoring the cell to prototrophy. Compared to pMTL-YN1 and pMTL-YN2, plasmids pMTL-YN1C and pMTL-YN2C have an additional segment of DNA inserted between the left-hand homology arm (LHA) and the right-hand homology arm (RHA) which carries: a transcriptional terminator (T1) of the ferredoxin gene of Clostridium pasteurianum; a copy of the lacZ’ gene encoding the alpha fragment of the E.coli β-galactosidase, and; a multiple cloning site (MCS) region derived from plasmid pMTL20 [3].
References
[1] Ng YK, Ehsaan M, Philip S, Collery MM, Janoir C, Collignon A, Cartman ST, Minton NP. Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles. PLoS One. 2013;8(2):e56051.
https://doi.org/10.1371/journal.pone.0056051
[2] Ehsaan M, Kuit W, Zhang Y, Cartman ST, Heap JT, Winzer K, Minton NP. Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824. Biotechnol Biofuels. 2016;9:4.
https://doi.org/10.1186/s13068-015-0410-0
[3]Chambers SP, Prior SE, Barstow DA, Minton NP. The pMTL nic– cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 1988; 68: 139–149.
https://doi.org/10.1016/0378-1119(88)90606-3