An allele-coupled exchange (ACE) vector system to enable rapid gene knock-in (KI). Rapid genome insertion of DNA can be achieved by utilising a mutant pyrE allele with genomic insertion concomitant with the restoration of a functional pyrE gene. This method may be used for gene complementation studies or the insertion of application specific modules.
These KI vectors 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 pyrE allele and DNA insertion at the pyrE 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 by uracil prototrophy restoration.
 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.