Last data update: 24 January 2024 16:39 CET
Plasmid name: pT74DsbA (LMBP 3058)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner non-core plasmid |
| Depositor's sequence: | pt74dsba.gb |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Escherichia coli gene facilitating disulfide bond formation (dsbA) |
| Promoter: | Phage T7 gene 10 promoter (T7g10) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli gene facilitating disulfide bond formation (dsbA) |
| Terminator: | - |
| Selection marker: | Ampicillin (amp) |
| Replicon: | Escherichia coli plasmid pMB1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pT74 |
| Further information: | The plasmid was constructed as follows: 1) Chromosomal DNA of the E. coli K12 lysogenic strain LMD52.30 (Strain number 2578) was digested with HindIII and ClaI, and the fragments obtained were subcloned into the HindIII-AccI vector fragment of pT74; 2) Subsequently, this library was screened with the synthetic oligonucleotide 5'-GCTGAATCCGCAGGGTATGG-3' corresponding to position 552-571 of the dsbA structural gene as published in Bardwell et al. pT74DsbA was succesfully used to produce high levels of enzymatically active DsbA in the E. coli BL21DE3 strain containing a controllable chromosomal T7 RNA polymerase gene, as well as in the E. coli B/r strain containing the helper plasmid pSCM22 carrying the T7 RNA polymerase information. This confirms that the cloned dsbA gene is preceded by its own ribosome binding site. For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3). Proceed as follows: first transform the expression strain with the auxiliary plasmid, make the transformants competent again, and then transform with the expression plasmid. The dsbA insert was verified by DNA restriction enzyme analysis as well as by DNA sequence analysis starting from the HindIII site located in the 5' flanking region up to approx. 150 bp of the dsbA coding region. Since this analysis does not cover the complete dsbA insert, different sources of nucleotide sequence information were used to reconstruct this plasmid: the sequence of the 5' flanking region of the dsbA gene (HindIII-RspXI fragment) was taken from the DNA sequence analysis results, although containing some unidentified nucleotides, and the sequence of the dsbA coding region (starting at the RspXI site) up to the SspI site was taken from the publication of Bardwell et al. As a result, we cannot exclude that some nucleotides in the present gene are different from the sequence as published by Bardwell et al. Due to the presence of some unidentified nucleotides in the 5' flanking region of the dsbA gene and since most of the nucleotide sequence of the 3' flanking region is not known, there is uncertainty as to the cutting frequency of the restriction enzymes indicated. |
| EMBL Accession number: | - |
| Latest sequence update: | 23/06/1994 |
| Authenticity test: | The plasmid still needs to be subjected to the authenticity test. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Dr K. De Sutter(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | - |
| Related plasmid reference: | Bardwell et al., Cell 67 (1991), 581-589 [PMID: 1934062] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 MC1061 |
| Host reference: | Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493] |
| Related host reference: | Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) |
| Cultivation temperature: | 37°C |
| Biosafety level: | L1 |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pT74DsbA (LMBP 3058) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter . |
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.