Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

1Introduction The ocean covering more than 70% of theearth’s surface represents a less explored environ-ment for microbial diversity(Yu etal., 2005). As agreat promising source for new natural productswhich have not been observed from terrestrial micro-o…

Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

Optimal conditions for protoplasts formation of marine Actinomycete sp. isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure (2 mg/cm3, 37 ℃, 40 min), and the concentration of sucrose in protoplast buffer was 0.4 mol/dm3 for keeping the balance of osmotic pressure. Using PEG-mediated protoplasts transformation, the transformation frequency was 89 transformants per microgramme of pIJ702. Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E. coli ET12567 (pUZ8002) using shuttle vectors pPM801, pPM803 and a(ψ)C31-derived integration vector pIJ8600 containing oriT and attP fragments. Transformation frequencies were 5.30×10-4±0.26×10-4, 8.92×10-4±0.19×10-4 and 6.38×10-5±0.41×10-5, respectively. Further, the heterologous expression of the allophycocyanin gene (apc) in the strain M048 was used to demonstrate this transformation system. SDS-PAGE and Western blot analysis confirmed the expression of recombinant APC (rAPC).