Clostridium Knock-In
Knock-In Vectors
Allele-coupled exchange (ACE) vectors enable the rapid knock-in (KI) of genes into the genome. It is most simply achieved by utilising a host that contains an auxotrophic mutant allele and an ACE plasmid carrying a corresponding wildtype allele. Successful delivery is selected on the basis of restoration of prototrophy, a consequence of replacement of the mutant allele with the functional wildtype gene. The most widely used allele is the pyrE gene, the inactivation of which leads to uracil auxotrophy [2]. Other markers include the genes argH, pheA and purD [3], which when inactivated lead to arginine, phenylalanine and purine (a cocktail of adenine, guanine and hypoxanthine) auxotrophy. ACE-mediated delivery of DNA to the genome may be used for gene complementation studies [1] or the insertion of application specific modules, such as the orthogonal expression system, TcdR [3].
KI ACE vectors typically comprise the catP antibiotic selection marker, a suitably replication defective Gram-positive replicon, Gram-negative replicon (if applicable) and homology arms to enable repair of the mutant allele and DNA insertion at the corresponding locus, eg., pyrE, argH, pheA and purD locus. The DNA to be inserted may be cloned between these regions of homology, with insertion selected by a pseudo-suicide single crossover event followed by selection for restoration of prototrophy.
Organisms
- Clostridium acetobutylicum
- Clostridium butyricum (coming soon)
- Clostridioides difficile
- Clostridium sporogenes (coming soon)
References
[1] Minton NP, Ehsaan M, Humphreys CM, Little GT, Baker J, Henstra AM, Liew F, Kelly ML, Sheng L, Schwarz K, Zhang Y. A roadmap for gene system development in Clostridium. Anaerobe. 2016;41:104-112.
https://doi.org/10.1016/j.anaerobe.2016.05.011
[2] Heap JT, Ehsaan M, Cooksley CM, Ng Y-K, Cartman ST, Winzer K, Minton NP. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res. 2012;40(8):e59.
https://doi.org/10.1093/nar/gkr1321
[3] Omorotionmwan BB, Wang H, Baker JP, Gizynski K, Yoo M, Akaluka C, Zhang Y, Minton NP. Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum. Front Bioeng Biotechnol. 2023;11:1218099.
https://doi.org/10.3389/fbioe.2023.1218099