Construction of the broad range cloning vector pHC60mCherry: Plant-Microbe interaction applications

GUSTAVO RAMIREZ; NICOLAS VOZZA; ANGELES ZORREGUIETA; MARCELO TOLMASKY

Anaheim, California

Conferencia; Experimental Biology (EB) Meeting; 2010

Here we present the construction of a wide range fluorescent protein expression vector that is compatible with other commonly used fluorescence conferring vectors in gram negative bacteria. Cloning the red autofluorescent protein "mCherry" in the wide range vector pHC60 has produced a useful tool for various molecular applications such as multiple fluorescent tagged microscopies. The destination vector is capable of constitutively expressing genes in various genera (Rhizobium, Sinorhizobium, ect.). Additionally, its toxin-antitoxin origin of replication makes it highly compatible with plasmid vectors with pBBR origins. The pHC60 vector originally contains a green fluorescent protein (GFP) sequence under its constitutive promoter. The GFP coding sequence was liberated from the plasmid by a digestion reaction with the restriction enzyme BglII. A subsequent double digestion with the restriction enzymes KpnI and XhoI allowed the pHC60 plasmid without GFP to become linear. The mCherry coding sequence was also liberated from the pJPO11 pCAGGS Chop-mCherry-WPR cassette by digestion with the same restriction enzymes. Compatible ends then facilitated a straight forward ligation reaction of the mCherry fragment with the linear pHC60V (GFP free) target vector. Additionally, we observed, by fluorescent microscopy, Rhizobium leguminosarum biovar viciae labeled with the new pHC60mCherry plasmid in free-living and symbiotically associated states. A wide range fluorescent protein expression vector that is compatible with other commonly used fluorescence conferring vectors can have various applications particularly in multiple fluorescence tagged microscopy studies.