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ccdA

Part:BBa_K196000:Design

Designed by: Laetitia Warny   Group: iGEM09_ULB-Brussels   (2009-08-11)
Revision as of 12:48, 12 August 2009 by Laetitia (Talk | contribs) (→‎Description)


CcdA antidote with the mob promoter (reverse)

Description

Stabilization system : Higher plasmid stability = More proteins Principle: In the StabyExpressTM system, the antidote gene (ccdA) is introduced in the plasmid DNA under the control of a weak constitutive promoter : the mob promoter, which doesn’t come from E. coli but from a broad host range plasmid (pBHR1) . On the other hand, the toxic gene (ccdB) is introduced in the chromosome of the bacteria, which can be furnished by DelphiGenetics. Expression of the poison gene is under the control of a promoter strongly repressed in the presence of the plasmid. When the plasmid is lost, the antidote is degraded and the production of the toxin is induced, causing cell death. Practically this means that when during the pre-induction phase bacteria are grown, 100% of the bacteria will carry the vector. If they lose the vector, they will not obtain a growth advantage, but will die. Upon induction 100% of the bacteria will start producing the recombinant protein leading to higher yields of the target protein and less background caused by unwanted proteins. For manufacturers of recombinant proteins this system offers a great benefit because it is an antibiotic free expression system. Therefore the manufactured protein will also be free of traces of antibiotics. For more information, please visit the Delphi Genetics [http://www.delphigenetics.com].



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

Initially, the ccdA sequence was introduced in the plasmid in the reversed way. In order to leave the possibility to use it in both ways, we also designed this part in the forward way [1].

Source

This part comes from the StabyTM plasmid, designed by Delphi Genetics [http://www.delphigenetics.com].

References

CEDRIC Y. SZPIRER AND MICHEL C. MILINKOVITCH, Separate-component-stabilization system for protein and DNA production without the use of antibiotics, BioTechniques 38:775-781 (May 2005)

SZPIRER ET AL., Mobilization Function of the pBHR1 Plasmid, a Derivative of the Broad-Host-Range Plasmid pBBR1, Journal of bacteriology, Mar. 2001, p. 2101–2110 Vol. 183, No. 6