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DNA Vector Systems

pBacTag Tagging Vectors

Epitope- and GFP-Tagging Integration Vectors for Bacillus subtilis

The six pBacTag vectors allow for translational fusions of two types of tagging sequences, epitope and localization tags, to the 3' end of any chromosomal gene of interest within the B. subtilis chromosome and of any other bacterial species not allowing for replication of pBacTag.

The transcriptional fusions are integrated into the respective chromosomal gene via homologous recombination encoding a tagged fusion protein. The epitope sequences (DYKDDDDK, c-Myc, and HA) allow detection of tagged proteins within the cell using commercially available antibodies. Furthermore, DYKDDDDK, c-Myc and HA tags can be used to purify the fusion proteins by affinity chromatography. The Green Fluorescent Protein (GFP) localization tags GFP+ (which produces enhanced fluorescence) YFP and CFP can be used to localize a protein to a specific compartment within the cell.

Features:

easy purification of fusion proteins produced in B. subtilis
allows detection of fusion proteins in immunoblotting experiments by commercially available antibodies
helpful for localization of target proteins in specific cell compartments
tags: DYKDDDDK (also known as FLAG® ), HA, c-Myc, GFP, YFP, CFP

The pBacTag vectors (Kaltwasser et al., 2002) are derivatives of pMUTIN2 (Vagner et al., 1998). First, the lacZ reporter gene of pMUTIN2 was replaced with a polylinker with several unique restriction enzyme sites. To ensure efficient termination of transcription immediately downstream of the hybrid gene, the trpA terminator of the Escherichia coli tryptophan operon was inserted into the Spe I -Sph I sites in such a way that the Sph I site was destroyed. The resulting plasmid served as a backbone for the insertion of the six tagging sequences.

The DNA sequences coding for the three different epitopes were ligated as complementary oligonucleotides into the Xma III and Spe I sites, resulting in pBacTag-DYKDDDDK, -cMyc, and -HA ; by this manipulation the Spe I site was destroyed in all three vectors. The codons for the different amino acids were designed on the basis of the codon usage table published for B. subtilis, and the open reading frames were terminated with two consecutive stop codons to ensure efficient termination of translation.

The coding regions for the GFP and its two variants were generated by PCR and flanked with Xma III and Spe I sites. As templates, the plasmids pMN402 (coding for GFP+, a variant of the wild-type gfp exhibiting increased fluorescence) and pSG1186 and pSG1187 (coding for cfp and yfp, respectively) were used, resulting in the three plasmids pBacTag-GFP+, -CFP, and -YFP. All six tagging vectors retain the unique restriction sites Kpn I, Eco 47III, Cla I, and Xma III for insertion of PCR fragments.

Vector Map of pBacTag (-DYKDDDDK, -cMyc and -HA)

P spac: P spac promoter
trpAt : TrpA terminator
ColE1 ori: ColE1 origin
Amp^r: ampicillin resistance
T₁T₂T₀: Lambda t₀ and rrnB t₁ , t₂ terminators
lacI: lacI gene (lac repressor)
Em^r: erythromycin resistance
tag: tag sequence

Vector Map pBacTag (-GFP, -CFP and -YFP)

For a detailed handbook as a pdf-file, please click on the product No.

ORDER INFORMATION, SHIPPING & STORAGE
order#	description	amount*
PBT001	pBacTag-DYKDDDDK vector DNA	5 µg
PBT002	pBacTag-cMyc vector DNA	5 µg
PBT003	pBacTag-HA vector DNA	5 µg
PBT004	pBacTag-GFP+ vector DNA	5 µg
PBT005	pBacTag-CFP vector DNA	5 µg
PBT006	pBacTag-YFP vector DNA	5 µg
Shipped at room temperature (RT). Lyophilized plasmid DNA can be stored at 4°C. Once the DNA has been dissolved in sterile water or buffer we recommend storage at -20°C.

FLAG® is registered trademark of Sigma-Aldrich Biotechnology, USA.

See also chapter 4 of our online catalog.

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