BCCM/GeneCorner Plasmid Collection
Department of Biomedical Molecular Biology
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Martine Vanhoucke (Martine.Vanhoucke@ugent.be)
Additional Database Information
Example of an entry from the BCCM_LMBP catalogue.
(This example is taken from BCCM/GeneCorner catalogue submitted to CABRI on June 20,2016)
||Mertens et al., Gene 164 (1995), 9-15 [PMID: 7590329]
||This plasmid was deposited by Dr N. Mertens and Prof. E. Remaut (Dept of Molecular Biology, Ghent University, Belgium).
PL promoter patent
||Escherichia coli K12 MC1061(lambda)
||Escherichia coli plasmid pMB1 origin
||Any Escherichia coli with a cI function
For PL driven expression use a strain with a cIts function (cI857).
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., Bio/Technology 13 (1995), 175-179) or e.g. BL21(DE3)[pcI857].
In both cases, proceed as follows: first transform auxiliary plasmid, make competent cells again and then transform the expression plasmid.
||Expression vector: general expression
||Phage lambda major leftward promoter (lambda PL)
Phage T7 gene 10 promoter (T7g10)
||Ribosome binding site (RBS) of the phage T7 gene 10 (T7g10)
||Phage T7 gene 10 terminator (T7g10)
||Start of the nucleotide sequence in the middle of the non-unique EcoRI site between the PL and the T7 promoter.
The construction of this plasmid is described in Mertens et al., Gene 164 (1995), 9-15.
pLT10T was designed for bacterial expression of heterologous genes after an ApaI digestion and blunting the 3' sticky ends, making the
ATG start codon on the plasmid accessible for blunt-end ligation to fragments encoding the mature coding sequence.
Ligation to other sites will result in the production of a fusion protein.
BamHI, BstXI, HindIII, MluI and SacI cannot be used for expression, since the HindIII site contains a termination codon in phase with
the ATG codon.
Other name of the plasmid is pPLT10T.
The EcoRI and XbaI expression sites are not unique (use 3-fragment ligation).
The lambda PL promoter is covered by the following patents issued to Biogen, Inc, Cambridge, MA, USA: U.S. Patent No. 4,874,702;
Canadian Patent No. 1,207,251; European Patent No. 41,767.
||E-AatII, E-ApaI, E-AsuI, E-BseRI, E-ClaI, E-EcoRI, E-KpnI, E-SmaI,
E-SphI, E-XbaI, E-XhoI
U-AatII, U-AccI, U-AlwNI, U-ApaBI, U-ApaI, U-AsuII, U-BamHI,
U-BcefI, U-BcgI, U-BciVI, U-BglI, U-BplI, U-Bpu10I, U-BsaAI,
U-BsaXI, U-BseRI, U-BsmI, U-BspLU11I, U-BsrBI, U-BstXI, U-ClaI,
U-DraII, U-DraIII, U-Eam1105I, U-EcoNI, U-Esp3I, U-GsuI, U-HindIII,
U-KpnI, U-MluI, U-MstI, U-NaeI, U-Pfl1108I, U-PstI, U-PvuI,
U-PvuII, U-SacI, U-SapI, U-ScaI, U-SgrAI, U-SmaI, U-SnaI,
U-SphI, U-SspI, U-StuI, U-StyI, U-Tth111I, U-XhoI, U-XmnI
Nucleotide sequence around the Shine-Dalgarno (SD) position of
the T7 gene 10 ribosome binding site:
------ ApaI AatII SphI XbaI XhoI
EcoRI KpnI SmaI ClaI HindIII BamHI
*: Start codon.
@: Termination codon.
Punctuation indicates reading frame.
How to make the ATG start codon accessible for blunt-end ligation:
5' ATG.GGC.C|CG 3'
3' TAC|CCG.G GC 5'
| ApaI digestion
5' ATG.GGC.C 3'
3' TAC 5'
| T4 DNA polymerase
5' ATG 3'
3' TAC 5'
|: Cleavage site of ApaI.
Punctuation indicates reading frame.
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