Fig. 3.

The three types of ACE integration vectors used to restore the pyrE allele to wildtype in the pyrE mutant host in which a mutation (Gene X) has been made by allelic exchange. Integration cassettes are modular and inserted between the SbfI and AscI sites of the pMTL80000 vectors. Each vector has a long (1200 bp) Right hand homology arm (RHA) and a shorter (300 bp) left homology arm (LHA). The latter is composed of the 3′-end of the pyrE gene, while former comprises the 1200 bp region of the chromosome from immediately downstream of the pyrE gene. In the case of the ACE Correction vector [1], the 300 bp and 1200 bp regions are in effect a continuous 1500 bp region of homology to the host chromosome in this region. In the case of the Complementation [2] and Overexpression [3] vectors, the two homology arms are separated by a region of DNA comprising a lacZ containing multiple cloning site (MCS) region and a downstream transcriptional terminator (FT). The Expression vector additionally contains a strong promoter (P_fdx) from the C. sporogenes ferredoxin gene immediately before the lacZ′ gene. Using the MCS, the complementation and expression plasmid variants allow delivery of a functional copy of a knocked-out gene (Gene X) under the control of its native promoter, for complementation studies, or under the control of the strong promoter of the C. spororgenes ferredoxin gene (P_fdx), to allow an assessment of the effect of overexpressing the gene. In every case, integration if the ACE plasmid is initially via the longer RHA. Subsequent plasmid excision via the LHA restores the pyrE allele to wildtype, allowing the former pyrE minus host to grow on minimal media lacking uracil.