Background/aims: The development of an electro-transformation system and the construction of shuttle plasmids for Actinobacillus actinomycetemcomitans have enhanced the molecular analysis of virulence factors. However, inefficient transformation is frequently encountered. This study investigated the efficiency of electro-transformation and expression of Green Fluorescent Protein (GFP) in 12 different A. actinomycetemcomitans strains. The influence of the plasmid vector, serotype, and phenotype were the major factors taken into consideration.
Material and methods: Twelve serotyped A. actinomycetemcomitans strains were independently electro-transformed with two different Escherichia coli-A. actinomycetemcomitans shuttle plasmids (pVT1303 and pVT1304), both containing an identical ltx-GFPmut2 gene construct but a different backbone (pDMG4 and pPK1, respectively). The transformation efficiency, transformation frequency, and electro-transformation survival rate were determined by culture techniques. GFP expression was observed at the colony level by fluorescence microscopy.
Results: All strains could be transformed with both plasmids. However, major differences were observed for the transformation efficiency, transformation frequency, and electro-transformation survival rate between strains. The data demonstrated that plasmid vector, serotype, and phenotype are key players for obtaining a successful transformation. An inverted relationship between the electro-transformation survival rate and tranformation frequency was also observed. GFP expression was also influenced by phenotype, serotype and plasmid vector.
Conclusions: The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent.