TITLE:
A New Tandem Gene Construction Method Involving a Cloning System Using Poxvirus DNA polymerase, and Its Application to Gene Expression
AUTHORS:
Tatsuro Shibui, Daisuke Sakaguchi, Hiroyoshi Hara
KEYWORDS:
Poxvirus DNA polymerase, Gene Cloning, Tandem Repeat, GFP, T7 RNA Polymerase
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.5 No.10,
September
25,
2014
ABSTRACT:
A simple method for constructing polymerized genes using only restriction
enzymes and commercially available cloning systems was established. In this
system, gel isolations or purifications of target genes after restriction
enzyme digestions or PCR amplifications, which often cause errors and mutations
in the target gene sequence, are not necessary. To verify the usefulness of
this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression
gene in Escherichia coli were
sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E.
coli showed an increasing trend in accordance with the copy numbers of the
gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining,
no expressed protein could be seen in E.
coli cells harboring plasmids that contained one or two copies of the gene.
However, expressed protein bands in E.
coli cells were clearly detected with 4 copies of the gene. In quantitative
analyses involving green fluorescence intensities per culture volume, the expression
level in E. coli with 16 copies of
the gene was 36.3-fold higher than that in E.
coli with one copy at 22 hours after induction.