Table 2 Primer pairs used in this study a
Primer pair | PCR Product/Description | Primer sequences |
|---|---|---|
P1: HVO_0665 preKO BamHI RV1 | Primers anneal ~500 bp upstream and downstream of hvo_0665; used with H26 genomic DNA as template to generate pJAM2819 (pre-deletion) by ligation into HindIII to BamHI sites of pTA131 | 5’- ATGAAGCTT AACGCGAGTCTCCTGTGGGCGCTCGG-3' |
P2: HVO_0665 HindIII preKO FW1 | 5’-ATTGGATCC GACGCGCGCACCTCGCCGTTC-3’ | |
P4: HVO_0665 3’-inverse | Used to generate plasmid pJAM2820 (Δhvo_0665) by inverse PCR using pJAM2819 as a template | 5’-TCCCGCGCCGGCCGACGACTGA-3’ |
P3: HVO_0665 5’-inverse | 5’-TCCGTCGCGTCGGTGAAGCCGTCGAACGACAT-3’ | |
P7: HVO_0665 700 bp confirm FW | Anneal ~700 bp upstream and downstream of hvo_0665; used to confirm Δhvo_0665 | 5’-GCTCGGCGGGGCGAACACG-3 |
P8: HVO_0665 700 bp confirm RV | 5’-GTGACCCACGAGACGACCCACGCG-3’ | |
P5: HVO_0665 NdeI | Used to screen for Δhvo_0665 and generate pJAM2821 (in trans complement of Δhvo_0665) | 5’-GGGCGGCATATGTCGTTCGACGGCTTCAC-3’ |
P6: HVO_0665 KpnI | 5’-TTGGTACCGTCGTCGGCCGGCGC-3’ | |
P7: C165A THI4 FW | Used to generate pJAM2822 for synthesis of the site-directed variant HvThi4 C165A | 5’- CGCGAACTCACGGCG GTCGACCCCATC-3’ |
P8: C165A THI4 RV | 5’- GATGGGGTCGACCGC CGTGAGTTCGCG-3’ |