Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.
For this demonstration, GFP-plasmid-harboring Escherichia coli cells were used and cultured in LB broth [0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 1% (w/v) NaCl with 0.1 μg/μL ampicillin and 1 mM IPTG].
The latter model was based on the demonstration that Y′ elements, which contained a weak origin of replication (Chan and Tye, 1983), could be propagated as extrachromosomal plasmids (Horowitz and Haber, 1985).
These observations are supported by the similarity of the replication genes, repABC, of those pairs of plasmids, as well as experimental demonstration of incompatibility between the plasmid pairs (Clark, Mattson, Garcia and Hynes, in preparation).
We have confirmed this result, including demonstration that the MmTKL1 plasmid is indeed present in the S. cerevisiae transformant (data not shown).
In this study we report the first demonstration of native BoNT-encoding plasmid transfer from an A3 subtype strain, an A4/bivalent B subtype strain and a nonproteolytic serotype B strain to a nontoxigenic mutant of a subtype A1 strain.
In any case, the demonstration that 99% of CEN plasmid segregations occur normally validates our conclusions.
In a demonstration of the approach, three AID-tagging plasmids were generated expressing GFP, CFP, or mCHERRY fluorescence.
As a proof-of-principle demonstration, the method of primer design was applied in colony PCR for identifying plasmid DNA deletion or insertion mutants after site-directed mutagenesis.
This is evidenced by the recent demonstration of the presence of the Swedish C trachomatis variant with partial deletion of plasmid in many European countries 18 as well as another distinct variant strain that could not be detected by plasmid based nucleic acid amplification tests.
