Open AccessProduction of green fluorescent protein by the methylotrophic bacterium Methylobacterium extorquens
Author(s) -
Figueira Marianne M,
Laramée Louise,
Murrell J.Colin,
Groleau Denis,
Miguez Carlos B
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09423.x
Subject(s) - green fluorescent protein , electroporation , plasmid , methylobacterium , promoter , biology , expression vector , chemistry , microbiology and biotechnology , bacteria , gene , gene expression , recombinant dna , biochemistry , genetics , 16s ribosomal rna
The production of green fluorescent protein (GFP) in Methylobacterium extorquens was studied by creating four different constructs using pJB3KmD, pRK310 and pVK101 vectors, as well as p Lac and soluble methane monooxygenase (sMMO) promoters. Plasmids were introduced into the cells by electroporation. Expression of GFP by selected clones was evaluated by growing cells in complex or defined media. The use of pRK310 as an expression vector containing the lacZ promoter resulted in a 100‐fold increase of GFP production when compared to cells containing the p Lac ‐GFP‐pJB3KmD construct. Higher production of GFP was observed also in cells containing p Lac ‐GFP‐pRK310 and p mmoX ‐GFP‐pVK101 constructs. While the transcriptional regulation of the smmo gene in Methylosinus trichosporium OB3b is known to be copper‐dependent, expression of GFP by M. extorquens clones harboring p mmoX ‐promoters was not strongly controlled by the presence of copper in the medium. The production of GFP was generally constant throughout the growth of M. extorquens carrying the p Lac ‐GFP‐pRK310 construct. GFP yields varied between 850 and 1000 μg of GFP g biomass −1 . However, the yield of GFP in cells carrying p mmoX ‐GFP‐pVK101 was somewhat reduced after the mid‐exponential phase of growth.