Premium
Molecular Cloning of Green Fluorescent Protein in Escherichia coli
Author(s) -
Shoneye Temitope,
Lee Henry,
Church George
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb854
Subject(s) - green fluorescent protein , plasmid , microbiology and biotechnology , escherichia coli , biology , cloning (programming) , molecular cloning , gene , multiple cloning site , expression vector , transformation (genetics) , gene expression , genetics , recombinant dna , computer science , programming language
Molecular cloning of green fluorescent protein (GFP) can be expressed as a functional transgene and enables new avenues of investigation, providing an easily detectable phenotype that can be used for studies. We hypothesized GFP can be expressed in E. coli cells. We sought to test the functional expression of a gene coding for agglutinin, from Vibrio natriegens . We tested for the functional expression of GFP from Aequorea victoria in E. coli because earlier work showed no positive results in the PCR or initial transformation reactions. We ran PCR on protein coding DNA (GFP), cloned GFP into plasmids and transformed the plasmids into E. coli cell by varying the number of templates, and increased the denaturing time from 10 to 20 sec. We also ran reactions with primers designed for blunt and cohesive ends cloning, and “touch‐down” PCR. We obtained a GFP sequence, designed primers and obtained correct products between 0.7 and 0.8 kb. We then digested the GFP product and the host vector with cohesive end cutters, KpnI and HindIII. Following ligation of the cohesive ends, we transformed the plasmids into chemically competent E. coli cells and tested the functional expression of GFP by measuring fluorescence. The results showed E. coli is able to functionally express GFP and that using the appropriate techniques, GFP expression in E coli is possible.