The markerless undA in-frame deletion mutant (ΔundA) contains a deletion of 762 base pairs (bp) in the undA gene. To obtain this deletion, the upstream and downstream regions of undA were amplified by PCR with the two primer pairs M1-undA/M2QC-undA (977 bp) and M3QC-undA/M4-undA (1,146 bp) respectively. Amplicons were used in overlap-PCR and re-amplified using M1-undA/M4-undA primers. The deleted undA gene construction was introduced into pAKE604 suicide vector (El-Sayed et al., 2001 (link)) digested by SmaI via blunt-ended ligation using T4 DNA ligase (NEB) and transformed in E. coli top 10 strain (Thermo Fisher Scientific). This construction was verified by Sanger-sequencing (Genewiz, Germany) and introduced into E. coli S17.1 strain (Simon et al., 1983 (link)). This plasmid was transferred in MFE01 by biparental mating as described by Bouteiller et al. (2020) (link), and double recombination event was selected. The in frame undA deletion mutant was checked by PCR analysis and DNA sequencing.
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