Qiaquick spin column kit

To construct an in-frame nonpolar tfoX mutant, the target vector pK18-tfoX was built as follows: first, the 653-bp upstream homologous region and the 656-bp downstream homologous region of the in-situ tfoX gene were amplified using the primers P5/P6 and P7/P8 from the genomic DNA of G. parasuis SC1401, then the 935-bp kanamycin-resistant cassette (Kan^R, aminoglycoside phosphotransferase Aph (3′)-IIa gene) was amplified by PCR from pKD4 using the primers P9/P10. Subsequently, these three fragments were purified using a Qiaquick spin column kit (Qiagen, Hilden, Germany) and were ligated into the linearized vector pK18 mobSacB. Similarly, the tfoX gene of SC1401 and SH0165 were amplified, and the four fragments were ligated into pK18 mobSacB to construct the target vector pK18-SC1401tfoX-Kan and pK18-SH0165tfoX-Kan. Finally, the resulting plasmids were transformed into the G. parasuis strain SC1401 using a natural transformation technique. Transformant bacteria were incubated for 36 h. The resultant kanamycin-resistant transformants were cultured on a large scale in TSB++/Kan for further identification by PCR.

The EspP2 deletion strain was constructed and screened as previously described [46 (link)]. Briefly, primers P1/P2 and P3/P4 were used to amplify, respectively, the 620-bp upstream and the 611-bp downstream homologous regions of EspP2 from G. parasuis SC1401 genomic DNA. Next, primers P5/P6 were used to amplify a 935-bp kanamycin resistance cassette from pKD4. Subsequently, the three fragments were purified using a Qiaquick spin column kit (Qiagen, Hilden, Germany) and ligated into restriction sites BamH I and Xho I of linearized pK18mobSacB, resulting in the vector pK18-EspP2. A natural transformation method was used to convert pK18-EspP2 into G. parasuis SC1401, as previously described [46 (link)]. After 36 h incubation, the resultant kanamycin-resistant transformants were grown to large-scale culture in TSB++ supplemented with Kan, in order to be further identified by PCR. Of note, we previously tested the natural transformation efficiency of several standard and local strains of G. parasuis and found that SC1401 had the highest efficiency. We therefore chose it as the wild-type strain for this study and from there constructed the EspP2 deletion strain.