Bacillus subtilis Epitope- and Fluorescence-Tagging Integration Vectors
pBacTag Tagging Vectors
Fig. 1 Chromosomal integration of pBacTag-GFP+: The 3’ end of orf2 (orf2’) was ligated into the multiple cloning site of the pBacTag-GFP vector. The orf2 belongs to an operon containing a total of three genes (orf1, orf2, and orf3). The pBacTag-GFP+ vector (with orf2’) is integrated into the chromosome of B. subtilis via homologous recombination of both orf2’ sites, by a single crossing over event. Now, the complete orf2 is fused to the gfp+ gene and can be transcribed from the native promoter localized upstream of orf1. Transcription of the tagged gene is terminated at the trpA terminator downstream of gfp+. Now, orf3 is no more transcribed from the native promoter of the operon. Instead, its transcription can be ensured from the Pspac promoter by adding IPTG. Broken arrows denote the promoters of the operon and Pspac. Promoters of lacI and the resistance genes are not depicted. The lollipop strands denote the trpA and the three lambda terminators (T1,T2,T0).
- Directed functional studies of B. subtilis genes by chromosomal integration
- The gene of interest can be selectively inactivated within the chromosome allowing phenotypic studies of the resulting mutant
- Alternatively, the gene of interest can be expressed chromosomally as translational fusion protein with an N-terminal epitope or localization tag
- After inactivation or tagging, genes localized directly downstream of the gene of interest can be controlled by an IPTG inducible promoter to ensure their expression
- Epitope tags (FLAG®, cMyc, and HA) can be used for selective purification of fusion proteins and for their detection by commercially available antibodies
- Localization tags (GFP+, CFP and YFP) are helpful for localization studies of target proteins
- The pBacTag Tagging Vectors enable the directed functional analysis of genes. Tagging or inactivation of target genes is achieved by chromosomal integration of a pBacTag Vector via homologous recombination.
- The mechanism of tagging a gene of interest with a pBacTag Tagging Vector is illustrated with pBacTag-GFP+ as example (Fig. 1). This vector can be used for creating a GFP+ fusion protein of any chromosomally located gene of interest, by fusing a gfp+ tag to the chosen gene. In this example the gene of interest is named orf2. It is part of an operon, including three genes in total (orf1, orf2, and orf3). For getting the gfp+ tag fused to orf2, the 3’ part of the gene (orf2’) has to be inserted into the multiple cloning site of the pBacTag-GFP+ vector. After transforming B. subtilis cells with this construct, chromosomal integration of the vector is achieved by selecting for cells with resistance against erythromycin. The integration is facilitated by homologous recombination of both orf2’ copies (one copy being within the plasmid, the other one within the chromosomal DNA). The mechanism of how the pBacTag-GFP+ vector (with orf2’) is integrated into the genome, is displayed in Figure 1.
- After vector integration, the complete orf2 is fused to the gfp+ gene and can be transcribed from the native promoter located upstream of orf1. Transcription of the tagged gene is terminated at the trpA terminator downstream of gfp+. orf3 (formerly within the operon) is no more transcribed from its native promoter. Instead, its transcription is driven by the IPTG inducible promoter Pspac.
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|PBT001||pBacTag-DYKDDDDK vector DNA* also known as FLAG Tag. FLAG is a registered Trademark by Sigma-Aldrich Co.||5 µg||328,00|
|PBT002||pBacTag-cMyc vector DNA||5 µg||328,00|
|PBT003||pBacTag-HA vector DNA||5 µg||328,00|
|PBT004||pBacTag-GFP+ vector DNA||5 µg||328,00|
|PBT005||pBacTag-CFP vector DNA||5 µg||328,00|
|PBT006||pBacTag-YFP vector DNA||5 µg||328,00|
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Vector maps and Sequences
- Kaltwasser M, Wiegert T, Schumann W.: Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis. Appl Environ Microbiol. 2002 May;68(5):2624-8. Pubmed
- Vagner V, Dervyn E, Ehrlich SD.: A vector for systematic gene inactivation in Bacillus subtilis. Microbiology. 1998 Nov;144 ( Pt 11):3097-104. Pubmed