Composite

Part:BBa_K1937010:Design

Designed by: Oumnia KARIM   Group: iGEM16_Toulouse_France   (2016-10-14)
Revision as of 22:15, 14 October 2016 by Oumniak (Talk | contribs) (→‎Design Notes)


Epsilon/MazF toxin and antitoxin in pSBBS0K-mini


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 257
    Illegal suffix found in sequence at 285
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 257
    Illegal NheI site found at 279
    Illegal SpeI site found at 286
    Illegal PstI site found at 300
    Illegal NotI site found at 263
    Illegal NotI site found at 293
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 257
    Illegal BglII site found at 4814
    Illegal BamHI site found at 330
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 257
    Illegal suffix found in sequence at 286
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 257
    Illegal XbaI site found at 272
    Illegal SpeI site found at 286
    Illegal PstI site found at 300
    Illegal NgoMIV site found at 1877
    Illegal AgeI site found at 4425
    Illegal AgeI site found at 5387
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 55
    Illegal BsaI.rc site found at 1701
    Illegal BsaI.rc site found at 3140
    Illegal BsaI.rc site found at 5656
    Illegal SapI site found at 618
    Illegal SapI.rc site found at 4638


Design Notes

Epsilon and MazF are under the control of the promoter Pveg (constitutive promotor for B. subtilis) followed by a strong RBS. A theophylline-dependent riboswitch was used to prevent the MazE toxin expression during the cloning steps in presence of theophylline (Topp and Gallivan, 2008). Indeed, with an expressed toxin, it would be impossible to clone the Biobrick in E. coli.SacII and KpnI are the restriction sites placed respectively before and after the riboswitch so that this fragment can easily be replaced depending on the control we want to apply to the bacteria. The construction was created by integrating the EcorI-PstI fragment from BBa_K1937009 in the pSBBS0K-miniplasmid (BBa_K1937001).


BBa K1937010-map.png

Source

The gene Epsilon codes for a toxin countered by the anti-toxin Zeta (Zielenkiewicz and Cegłowski, 2005 ; Mutschler et al., 2011). The gene MazF encodes the anti-toxin for the MazE toxin (Bravo et al., 1987, Zhang et al., 2005, Wang et al., 2013).

References