A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 69

9. OneShot Top10 Escherichia coli (Invitrogen).
10. Luria-Bertani (LB) Medium and a 37°C incubator for culture of E. coli .
3. Methods
The following describes the construction of genetic manipulation plasmids
and methods for electro-transformation that were utilized to generate a
B. hermsii vtp mutant and a reconstituted isogenic strain. This system can be
customized for mutagenesis and reconstitution of other genes by designing
endonuclease-tagged primers specific to the particular target locus. In silico
construction of plasmids and confirmation of the restriction profile by restriction
fragment length polymorphism (RFLP) are recommended prior to synthesis of
the primers.
Complementation refers to a genetic manipulation event wherein a wild-
type copy of the mutant gene is introduced, leaving the original mutation
intact. This can be achieved by inserting a wild-type copy of the mutant
gene (and its promoter region) into the B. hermsii shuttle vector pBhSV-2.
When the shuttle vector is used for restoration of a mutant genotype, this
autonomously replicating plasmid may artificially increase the relative number
of gene copies and could result in abnormal regulation and synthesis of the
gene of interest. Alternatively, genetic reconstitution involves the replacement
of the mutagenized gene with wild-type copy at the wild-type locus.
3.1. Construction of Genetic Manipulation Plasmids
3.1.1. Cloning of the Target Locus into Vector pOK12
1. Identify specific target gene of interest within the genomic sequence.
2. Design primers to amplify the pOK12 amplicon containing the p15A origin +
kan-r cassette and the target gene with 900-2000 bp of flanking DNA on each
side. The primers should include restriction endonuclease sites (tags) that are not
found in the target locus or within pOK12. Choose restriction enzymes that have
a compatible buffering system ( see Note 6 for primer design and construction
details relevant to pOKvtp, and Fig. 1 for an illustration of this plasmid).
3. Prepare genomic DNA ( B. hermsii DAH 2E7 in this example) using the
QIAtissue kit (Qiagen) per the manufacturer's instructions. Other methods of
preparing genomic DNA are suitable. As differences in the genomic sequence
between strains is likely, it is most prudent to prepare genomic DNA from the
spirochetal strain that is intended to be the host strain for genetic manipulation.
4. Amplify the target locus and the pOK12 amplicon using Expand Long Template
PCR System Kit (Roche Diagnostics) per the manufacturer's instructions and
analyze products by agarose gel electrophoresis for correct size ( see Note 7 ).
Adjust the annealing temperature and extension time to reduce non-specific
background products that could interfere with the cloning.