Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis.
Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.
Kaltwasser
Marcus
M
Institute of Genetics, University of Bayreuth, D-95445 Bayreuth, Germany.
Wiegert
Thomas
T
Schumann
Wolfgang
W
D000906:Antibodies; D000918:Antibody Specificity; D001412:Bacillus subtilis / Q000235:genetics*; D000939:Epitopes / Q000032:analysis / Q000235:genetics / Q000276:immunology; D014644:Genetic Variation; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000939:Epitopes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins