Difference between revisions of "PGP1087"

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* ''B. subtilis'': no ori, select for erythromycin/lincomycin
 
* ''B. subtilis'': no ori, select for erythromycin/lincomycin
  
* based pMUTIN-FLAG
+
* based on pMUTIN-FLAG
  
 
'''Hints for the usage'''
 
'''Hints for the usage'''

Revision as of 15:31, 31 October 2011

pGP1087: click to enlarge

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 references

Diethmaier C, Pietack N, Gunka K, Wrede C, Lehnik-Habrink M, Herzberg C, Hübner S, Stülke J    
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