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. 2019 Aug 20;9:287. doi: 10.3389/fcimb.2019.00287

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This work is authored by Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter and Rosa on behalf of the U.S. Government and, as regards Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter, Rosa, and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply

This work is authored by Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter and Rosa on behalf of the U.S. Government and, as regards Hillman, Stewart, Strnad, Stone, Starr, Carmody, Evans, Carracoi, Wachter, Rosa, and the U.S. Government, is not subject to copyright protection in the United States. Foreign and other copyrights may apply. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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PCR confirmation of genetically modified B. burgdorferi spirochetes. (A) Diagram of relevant genetic loci and corresponding agarose gel image of PCR amplicons. Native loci are shown in red, the kanamycin-resistance cassette (flgBp aph1) is in blue, and relevant portions of the shuttle vectors SV-TC and SV-2xTC are shown in green. Small arrows above diagram indicate oligonucleotides K and J used for PCR confirmation of the ospDTC gene in B. burgdorferi transformants. The endogenous plasmid (lp38) locus in the parental B. burgdorferi strains (A3ΔOspD 109 and B31A34) does not amplify, while the transforming shuttle vector DNA serves as a positive control (+). (B) PCR amplification of the chromosomal la7TC region utilizing primers P and Q (small arrows above diagram). Native loci are shown in red, while DNA introduced by allelic exchange, including the kanamycin-resistance cassette (flgBp aph1), is shown in green. The parental B. burgdorferi strain (B31 A3) serves as a negative control, while the transforming plasmid DNA acts as a positive control.