Difference between revisions of "PGP1087"
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'''General comments''' | '''General comments''' | ||
| − | Integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor | + | * Integrative [[plasmid]] for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor, allows express of the downstream gene by the p(spac) promoter |
| − | * ''E.coli'': | + | * constructed in [[Jörg Stülke]]'s lab (C. Diethmaier) |
| + | |||
| + | * ''E.coli'': low copy plasmid, select for ampicillin | ||
* ''B. subtilis'': no ori, select for erythromycin/lincomycin | * ''B. subtilis'': no ori, select for erythromycin/lincomycin | ||
| + | |||
| + | * based on pMUTIN-FLAG | ||
'''Hints for the usage''' | '''Hints for the usage''' | ||
* amplify the last 300bp of your gene of interest, | * amplify the last 300bp of your gene of interest, | ||
| − | * skip the | + | * skip the stop codon of the gene in the reverse primer |
* take care that the integration is in-frame | * take care that the integration is in-frame | ||
'''Antibody''' | '''Antibody''' | ||
*dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it) | *dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it) | ||
| + | |||
| + | =The reference= | ||
| + | <pubmed>21856853</pubmed> | ||
| + | == Return to [[Plasmids]] == | ||
Latest revision as of 18:07, 2 August 2016
General comments
- Integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor, allows express of the downstream gene by the p(spac) promoter
- constructed in Jörg Stülke's lab (C. Diethmaier)
- E.coli: low copy plasmid, select for ampicillin
- B. subtilis: no ori, select for erythromycin/lincomycin
- based on pMUTIN-FLAG
Hints for the usage
- amplify the last 300bp of your gene of interest,
- skip the stop codon of the gene in the reverse primer
- take care that the integration is in-frame
Antibody
- dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it)
The reference
Christine Diethmaier, Nico Pietack, Katrin Gunka, Christoph Wrede, Martin Lehnik-Habrink, Christina Herzberg, Sebastian Hübner, Jörg Stülke
A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation.
J Bacteriol: 2011, 193(21);5997-6007
[PubMed:21856853]
[WorldCat.org]
[DOI]
(I p)
