Difference between revisions of "Part:BBa K1937010:Design"
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Revision as of 22:15, 14 October 2016
Epsilon/MazF toxin and antitoxin in pSBBS0K-mini
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 257
Illegal suffix found in sequence at 285 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 257
Illegal BglII site found at 4814
Illegal BamHI site found at 330 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 257
Illegal suffix found in sequence at 286 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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).
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).