Table 1 Bacterial strains, plasmids and primers used in this study
Bacterial stain | Characteristics | Sources |
|---|---|---|
HuN001 | Wild-type P. multocida strain, capsular type A | Our laboratory |
PM-ΔpetL | petL-deletion strain of HuN001 | This study |
PM-CpetL | petL-complementary strains | This study |
KP01 | Wild-type K. pneumoniae strain from pigs | Our laboratory |
KP02 | Wild-type K. pneumoniae strain from pigs | Our laboratory |
DH5α | E. coli strain | YIBAI |
MG1655 | E. coli K12 strain | ANGYUBIO |
Plasmids | ||
pSHK5Ts-NgAgo | NgAgo and RBS gene insert between the Kan promoter and Kanr gene of temperature-sensitive plasmid pSHK5Ts, can express NgAgo protein;Kanr | Gifted by Anding Zhang |
pPBA1101 | E. coli-P. multocida shuttle vector, Kanr | Gifted by John Boyce |
pMD19-T | Cloning vector; Ampr | TAKARA |
pSHK5Ts-ΔpetL | petL upstream and downstream fragment amplified from HuN001 ligated into pSHK5Ts-NgAgo; Kanr | This study |
pPBA1101-petL | pPBA1101 containing petL; Kanr | This study |
pMD19-T-petL | pMD19-T containing petL; Ampr | This study |
Primers | ||
Primers | Sequences (5’ ~ 3’) | Product size |
petL-F | ATGAAAAAATTACGATTTCGTT | petL full length (1,632 bp) |
petL-R | CTAGCGTATGCGACAAC | |
petL-L-RH-F | CGCGGATCCAATTCGATGCCTTTCTCATCAGT | petL upstream fragment (821 bp) |
petL-L-RH-R | TAGGAGTTTTGTTGTTATAGGACATTGAATAAAAGCCAAGTAAAG | |
petL-R-RH-F | TTTATTCAATGTCCTATAACAACAAAACTCCTAAAAAAATCGACT | petL downstream fragment (890 bp) |
petL-R-RH-R | CGGGGTACCAAGGTATTGGGCAACAGTTAGT | |
petL-inner-JD-F | AATCTGGCAGAAGGTTCA | Identification of petL deletion (438 bp) |
petL-inner-JD-R | GGTGGTTTGTCGCTCATA | |
petL-outer-JD-F | ACTGGATTTACGCCGTTTGTTTGA | Identification the recombinant of petL (wild type: 4,100 bp; mutant type: 2,468 bp) |
petL-oute-JD-F | TCCACCGAAAACACAATCTTGCCC | |
100-q-petL-F | CCGTTATTAGCAAATCGGGGCG | qPCR for petL |
100-q-petL-R | ATCTGGAAAACATACAGGGGAC | |
q-aroA-F | ACACCGAACAAGGAAGGCAA | qPCR for aroA |
q-aroA-R | TCTGCGCCAGCTTGACATAA | |
pPBA-HindIII-F | CCCAAGCTTGATGAAAAAATTACGATTTCGTTGGTCTTTA (HindIII) | petL full length (1,632 bp) |
pPBA-Sac I-R | CCCGAGCTCCTAGCGTATGCGACAACGA (SacI) | |
pMD19-Sal I-F | ACGCGTCGACGATGAAAAAATTACGATTTCGTTGGTCCTTA (SalI) | petL full length (1,632 bp) |
pMD19-Sac I-R | CCCGAGCTCCTAGCGTATGCGACAACGA (SacI) | |