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PBT000 - Bacillus subtilis pBacTag Tagging Vectors, 5 µg

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pBacTag Vector



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The pBacTag Vectors are Bacillus subtilis epitope- and fluorescence-tagging integration vectors for the directed functional analysis of B. subtilis genes. All pBacTag tagging vectors are derivatives of pMutin vectors. pBacTag tagging vectors can replicate in E. coli but are unable to propagate in B. subtilis, enabling chromosomal integration with B. subtilis (and other bacterial species in which pBR322 based plasmids are not able to replicate). The IPTG inducible Pspac promoter allows controlled expression of genes that are located downstream of the target gene. For proper cloning, the vectors contain a multiple cloning site downstream of the Pspac with the unique restriction sites KpnI, Eco47III, ClaI and EagI. To ensure efficient termination of transcription of the hybrid gene, the vectors contain the trpA terminator of the E. coli tryptophan operon downstream of the tag.
 
Tagging or inactivation of target genes is achieved by chromosomal integration of a pBacTag Vector via homologous recombination. The gene of interest can be selectively inactivated within the chromosome, enabling phenotypic studies of the resulting mutant. Alternatively, the gene of interest can be expressed chromosomally as a 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. 
 
Six pBacTag tagging vectors are offered as either epitope tags or localization tags: 
 
  • Epitope tags (PBT001, PBT002, PBT003) - used for selective purification of fusion proteins and for their detection by commercially available antibodies. Since the tags are very short, protein function is usually not disturbed.
 
  • Localization tags (PBT004, PBT005, PBT006) - helpful for localization studies of target proteins. The fusion proteins that contain a fluorescing tag can be analyzed for their cell compartmental localization. The GFP+ tag, pBacTagGFP+ (PBT004), is an improved variant of the common GFP, which produces enhanced fluorescence.
 
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).
 

Features

  • Gene of interest can be selectively inactivated within the chromosome or expressed chromosomally as a translational fusion protein
  • Can replicate in E. coli, but unable to propagate in B. subtilis
  • IPTG inducible Pspac promoter allows controlled expression of genes that are located downstream of the target gene
  • Contains a multiple cloning site downstream of the Pspac 
  • Contains the trpA terminator of the E. coli tryptophan operon downstream of the tag to ensure efficient termination of transcription of the hybrid gene
  • Both epitope tags and localization tags are available
 

Applications

  • Directed functional studies of B. subtilis genes by chromosomal integration
 

Vectors

  • PBT001 - pBacTag-DYKDDDDK vector DNA (also known as FLAG Tag), 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
 

Literature

Publications