Integrity of replacement in reverse orientation (Primers 4 & 5 and 6 & 7).
To confirm integration of hph+trpC at the desired locus and in the correct orientation, a primer from within hph and within 200 bp of a flanking region used to guide insertion were used to amplify a diagnostic region of approximately 1.5 kb.
Assays for which both flanking primers and forward and reverse orientation SNP primers could be designed were considered successful and validated (Additional file 6).
The clpX coding region was cloned in the sense orientation using primers ClpX-PacI and ClpX-SwaI or antisense orientation using asClpX-PacI and asClpX-SwaI under the inducible tetracycline (tet) promoter in pLR52, a replicating E. coli - Mycobacterium shuttle vector (Tables S1 and S2).
We analyzed the structures of rrn operon-flanking regions in five other strains of P. gingivalis (TDC60, TDC117, TDC275, SU63, and GAI7802) using a set of orientation-specific primer pairs, and we found that all of the rrn operon-flanking regions of these five strains have the same structures as those of ATCC 33277 (Supplementary Fig. S3).
The promoter fragment was amplified from pJET1.2 vector in both orientations using primers with suitable attB sites attached to them and cloned into Gateway® entry vector pDONR221 (Invitrogen).
The gel profile indicates that in the case of both TA and cohesive ligations for 60S-L37 and 40S-L17, relatively more inserts were in M13-Forward (-20 -CDS III/ 3' primer orientation (Lane 1, 3, 5, 7) than in the M13-Reverse-CDS III/ 3' primer orientation (Lane 2, 4, 6, 8).
To obtain the O. pardalina region located between hsp70 genes found in inverse orientation, we used primer outward from the 5'-end of hsp70.
The analysis started from the primers clipped 16Sr RNA sequences; containing sequences where primers were detected and removed (allowing 2 mismatches) and turn into forward and reverse primer orientation which was later combined using the make.contigs command.
As determined by band intensity values, the percentages of recombinants in the M13-Forward primer orientation were around 88%and68%8% for TA ligated 60S-L37 and 40S-L17; while in case of cohesive ligations, they were around 73%and71%1%, respectively.
