PBT004 pBacTag-GFP+ vector BioVector NTCC质粒载体菌种细胞基因保藏中心

PBT004 pBacTag-GFP+ vector

Introduction
Gram-positive bacteria are well known for their contributions to agricultural, medical and
food biotechnology and for the production of recombinant proteins. Among them, Bacillus
subtilis has been developed as an attractive host and model organism, because of
several reasons:
· Bacillus subtilis is non-pathogenic and awarded GRAS (generally regarded as safe)
status from the US Food and Drug Administration.
· There is no significant bias in codon usage.
· It is capable of secreting high levels of functional proteins directly into the culture
medium. At present, about 60% of the commercially available enzymes are produced
by Bacillus species.
· A large body of information on B. subtilis is already available, greatly facilitating
fundamental research experiments or the construction of improved protein production
strains. The complete B. subtilis genome information is available in addition to many
data on transcription, translation, protein folding, secretion mechanisms and gene
manipulation results.
However, the functions of the about 4,100 B. subtilis genes identified, are still
incompletely clarified. The pBacTag Tagging system has been developed to disburden
further functional studies. On this, the pBacTag Tagging Vectors enable the directed
functional analysis of genes by two different modes of action:
· Specific inactivation of genes of interest within the chromosome (followed by
phenotypical analysis)
· Chromosomal expression of the gene of interest as translational fusion with an
epitope or localization tag fused to the 3’-end (for selective protein purification,
detection by commercially available antibodies or for localization studies).
The tagging or inactivation of the target gene is achieved by chromosomal integration of
the pBacTag Tagging Vector into the B. subtilis chromosome by homologous
recombination.
2 The pBacTag Tagging Vectors
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. All pBacTag Tagging Vectors are derivatives of
pMutin vectors (Vagner et al., 1998; Kaltwasser et al., 2002) with the following properties:
· pBacTag Tagging Vectors are able to replicate in E. coli, but unable to propagate in
B. subtilis. The latter enables chromosomal integration with B. subtilis (and other
bacterial species, in which pBR322 based plasmids are not able to replicate), by
homologous recombination, using the erythromycin-resistance gene as selection
marker. For propagation in E. coli, the b-lactamase gene can be used for selection
purpose, causing resistance against ampicillin.
· The IPTG inducible Pspac promoter allows, after chromosomal integration, the
controlled expression of genes that are located downstream of the target gene. This
s important, because most of B. subtilis genes are organized in multicistronic units,
and downstream genes within the same operon may be separated from their native
promoter by the integration event. The Pspac is therefore an indispensable tool to
avoid polar effects from expression changes of downstream genes.
· For proper cloning, the vectors contain a multiple cloning site downstream of the
Pspac with the following 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.
· The terminators t1t2t0 (t1 and t2 of the E. coli rnb operon and the lambda terminator
lt0 downstream of the erythromycin resistance gene take care for proper RNA
polymerase termination at this place and prevent any “read through” to genes
downstream of the Pspac.
· Three of the tagging vectors - pBacTag-DYKDDDK (also known as FLAG®),
pBacTag-cMyc and pBacTag-HA – allow the expression of epitope tagged fusion
proteins. These proteins can be detected in immunoblotting experiments by using
commercially available antibodies against the respective tag. The fusion proteins can
also easily being purified using the tag in affinity chromatography. Since the tags are
very short (FLAG®: 7 aa, cMyc: 10 aa, HA (hemagglutinin): 9 aa) protein function is
usually not disturbed.
· Localization tags can be fused to the protein of interest, using pBacTag-GFP+,
pBacTag-YFP and pBacTag-CFP. The fusion proteins containing a fluorescing tag
can be analyzed for their cell compartmental localization. The GFP+ tag (pBacTagGFP+) is an improved variant of the common GFP, which produces enhanced
fluorescence.
2.1 Mechanism of pBacTag Tagging Vectors
The mechanism of pBacTag Tagging Vectors is illustrated with pBacTag-GFP+ as
example. This vector can be used for creating a GFP+ fusion protein from 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 the 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
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 ensured by the IPTG inducible promoter Pspac

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