Protein expression from an Escherichia coli/Bacillus subtilis multifunctional shuttle plasmid with synthetic promoter sequences.
A plasmid shuttle vector (pSP10) was designed and constructed to simplify screening of cloned DNA and to facilitate expression of the protein products. The plasmid contained the following features: (i) a selection gene, chloramphenicol acetyltransferase; (ii) an indicator gene encoding beta-galactosidase for visual identification of colonies containing DNA inserts; (iii) a cloning region immediately upstream from the indicator gene; (iv) origins of replication recognized by both Escherichia coli and Bacillus subtilis; and (v) a synthetic DNA expression control sequence, including -35 and -10 regions, ribosomal binding site, and transcriptional and translational start sites. The promoter region is a synthetic consensus sequence derived from published B. subtilis promoters. The plasmid has been shown to replicate actively in E. coli and B. subtilis and to confer chloramphenicol resistance to both hosts. DNA inserted at the cloning region inactivates the indicator gene, resulting in white colonies on 5'-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside plates. beta-Galactosidase has been expressed from pSP10 in both E. coli and B. subtilis. A comparison was made of the expression levels of beta-galactosidase from the same plasmid which had been modified to contain: (i) the synthetic control region, (ii) no promoter region, (iii) the synthetic control region cloned in the opposite orientation, or (iv) the tac promoter.
Trumble
W R
WR
Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83843.
Sherf
B A
BA
Reasoner
J L
JL
Seward
P D
PD
Denovan
B A
BA
Douthart
R J
RJ
West
J W
JW
D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular / Q000379:methods*; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007613:Kanamycin Resistance; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase