Construction of undA Deletion Mutant

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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Dupont C.A., Bourigault Y., Osmond T., Nier M., Barbey C., Latour X., Konto-Ghiorghi Y., Verdon J, & Merieau A. (2023). Pseudomonas fluorescens MFE01 uses 1-undecene as aerial communication molecule. Frontiers in Microbiology, 14, 1264801.

Publication 2023

T4 DNA ligase E. coli top 10 strain Sanger-sequencing

Deletion E coli Frame Gene Ligation Plasmid Primers Recombination Smai Strain T4 dna ligase Vector

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Other organizations : Écologie et Biologie des Interactions

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Variable analysis

independent variables

Deletion of 762 base pairs (bp) in the undA gene

dependent variables

Presence of the undA deletion mutation

control variables

Amplification of the upstream and downstream regions of undA gene with specific primer pairs
Overlap-PCR to obtain the deleted undA gene construct
Cloning of the deleted undA gene construct into the pAKE604 suicide vector
Transformation of the construct into E. coli Top10 strain
Verification of the construct by Sanger sequencing
Introduction of the construct into E. coli S17.1 strain
Transfer of the plasmid to MFE01 strain by biparental mating
Selection of the double recombination event
Confirmation of the undA deletion mutant by PCR analysis and DNA sequencing

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