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LMBP plasmid details

Last data update: 24 November 2020 04:18 CET

Plasmid name: pPLT10igaABT (LMBP 3242)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p3242.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Neisseria gonorrhoeae NCTC8375 gene encoding IgA1 protease (iga); α and β domains (igaαβ)
Promoter: Phage λ major leftward promoter (λ PL)
Phage T7 gene 10 promoter (T7g10)
Ribosome
binding site:
Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)
Terminator: Phage T7 gene 10 terminator (T7g10)
Selection marker: Ampicillin (amp)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli; use strains with a cI function, cIts for PL controlled expression
Parental clone: pLT10T; pMc514igam2
Further information: The plasmid was constructed by inserting the ScaI-HindIII fragment from pMc514igam2, encoding the α and β domains of the Neisseria gonorrhoeae NCTC8375 IgA1 protease, into the ApaI (blunted with T4 DNA polymerase) - HindIII opened pLT10T vector.
This is an expression plasmid for the IgAαβ domain of the IgA1 protease under control of the PL or the T7 promoter.
For T7 driven expression use a strain containing a controllable T7 RNA polymerase gene, as well as a cI function: preferably a pT7POL plasmid (Mertens et al., Biotechnology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857]. Proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
To reconstruct the nucleotide sequence of this plasmid, the sequence of the IgA1 protease gene from Neisseria gonorrhoeae MS11 corresponds with the EMBL Nucleotide Sequence Database (Release 36; Accession number X04835). However, the cloned IgA1 protease gene was derived from Neisseria gonorrhoeae NCTC8375 and differs in several nucleotides. As such, there is uncertainty as to the cutting frequency of the restriction enzymes indicated.
Other name of the plasmid is pLT10tIgAαβ.
EMBL Accession number: -
Latest sequence update: 27/07/1995
Sequence detail:
Nucleotide sequence at the start of the igaAB domain.



                       Met Thr Pro Ala Ala
5' TTAAGAAGGAGATATACAT ATG.ACT.CCT.GCC.GCA 3'
        ------           -----
          SD          ApaI/ScaI fusion


SD: Shine-Dalgarno position.
Punctuation indicates reading frame.
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 I. Poelaert(1) (2) and Prof. Dr E. Remaut(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: PhD thesis Irmgard Poelaert (1995)
Restricted distribution: - BCCM MTA
Distributed as: H/P active culture or plasmid DNA
Host for distribution: Escherichia coli K12 MC1061(λ)
Host reference: Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
Related host reference: Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
Cultivation medium: LB-Lennox + ampicillin (100 μg/ml)
Cultivation temperature: 28°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

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.

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