Last data update: 28 November 2020 04:24 CET
Plasmid name: pMc519SAigaAB (LMBP 3233)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Escherichia coli alkaline phosphatase A gene (phoA); signal sequence
Streptomyces avidinii streptavidin gene (SA); mutated mature sequence
Neisseria gonorrhoeae NCTC8375 gene encoding IgA1 protease (iga); α and β domains (igaαβ)
|Promoter:||Escherichia coli hybrid tryptophan/lacUV5 promoter (tac)|
|Ribosome binding site (RBS) of the Escherichia coli alkaline phosphatase A gene (phoA)|
|Terminator:||Phage fd terminator|
|Selection marker:||Chloramphenicol (cam)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli|
|Parental clone:||pMc519; pMc58SAm4; pMc514igam2|
|Further information:||The plasmid was constructed as follows: 1) The AccI (filled-in with Klenow DNA polymerase) - BamHI fragment, encoding the mature S. avidinii streptavidin gene (mutated at the position of the first codon and of the termination codon), was isolated from pMc58SAm4; 2) The ScaI-HindIII fragment, encoding the α and β domains of the Neisseria gonorrhoeae NCTC8375 IgA1 protease, was isolated from pMc514igam2; 3) The isolated fragments were subcloned into the HindIII-BamHI opened pUC18 vector, resulting in the intermediate construction pUC18SAIgAαβ; 4) The BamHI (filled-in with Klenow DNA polymerase) - HindIII fragment from pUC18SAIgAαβ, encoding the SA-igaαβ fusion gene, was then ligated to the KpnI (blunted with T4 DNA polymerase) - HindIII opened plasmid pMc519.
The signal sequence of the E. coli alkaline phosphatase gene (sphoA) starts with ATG, while in the wild type sequence the start codon is GTG.
Only 13 nucleotides upstream the ATG start codon of sphoA are derived from the original ribosome binding site of phoA.
pMc519SAigaAB allows expression of streptavidin fused to the Neisseria gonorrhoeae IgAαβ domain as a carrier molecule. The expression is under control of the tac promoter which is regulated by the lacI(q) repressor present in the bacterial genome or on a compatible plasmid (e.g. pDMI,1).
Streptavidin was functionally exposed at the E. coli surface, since intact SA-IgAαβ-expressing cells could specifically bind biotin and biotinylated alkaline phosphatase. These results illustrate the applicability of the IgAαβ domain of IgA protease as a vehicle and anchor to expose foreign polypeptides at the surface of E. coli.
The sequence of the streptavidin gene was taken from GENBANK (Release 55; Locus: STMSTRAVG).
After mutagenesis, use mutS strains for primary transformation (e.g. sup(-) strain WK6mutS, sup(+) strain BMH71-18mutS); for segregation of possible mutants: sup(-) strains (e.g. WK6).
The nucleotide 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 fragment was derived from Neisseria gonorrhoeae NCTC8375 and may be different in several nucleotides. As such, there is uncertainty as to the cutting frequency of the restriction enzymes indicated.The presence of the lacI(q) repressor gene is necessary to control the activity of the tac promoter.
Other names of the plasmid are pMc519SAIgAαβ and pMc519SAIgAαβ.
|EMBL Accession number:||-|
|Latest sequence update:||29/06/1995|
Nucleotide sequence at the fusion between the streptavidin gene and the igaAB domain. AccI/ScaI fusion | streptavidin|alpha protein of the iga gene --->|---> Val Gln Gln Ser|Thr Pro Ala Ala Asn 5' GTT.CAG.CAG.TCG|ACT.CCT.GCC.GCA.AAC. 3' ^ | -------- HindII SalI
|Authenticity test:||The plasmid still needs to be subjected to the authenticity test.|
|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 RR1|
|Cultivation medium:||LB-Lennox + chloramphenicol (25 μg/ml)|
|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.