Part:BBa_K1321328

J-sRNA-331Bb

This construct constitutively expresses LuxR and expresses an sRNA and E. coli Hfq behind an AHL-inducible promoter, which leads to suppression of cellulose production in Komagataeibacter rhaeticus iGEM. The 5' of the sRNA is complementary to K. rhaeticus iGEM UGPase mRNA RBS region, and 3' has an E. coli Hfq binding region - upon expression, it complexes with UGPase mRNA, and leads to Hfq-mediated translational repression. This construct is a member of the Komagataeibacter genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332).

Komagataeibacter toolkit was designed to ease genetic engineering of cellulose-based biomaterials using the cellulose synthesizing bacterium Komagataeibacter rhaeticus iGEM (parts Bba_K1321305 and BBa_K1321306). As no registry parts had been tested in K. rhaeticus, the aim of this toolkit was to determine the parts usable in K. rhaeticus and to characterize them in this host. pSEVA331-Bb is a non-standard broad host range plasmid capable of replication in K. rhaeticus and E. coli (a shuttle vector) and was selected because the registry's standard plasmid backbone pSB1C3 can not be used for K. rhaeticus engineering.

NOTE: Because the registry's standard plasmid backbone pSB1C3 is not capable of replication in Komagataeibacter or Gluconacetobacter species, the Komagataeibacter genetic engineering toolkit is housed mainly in pSEVA331-Bb. pSEVA331-Bb is a non-standard backbone, which therefore can't be quality controlled by and maintained in the Registry. However, in order to make the Komagataeibacter toolkit available for the synthetic biology community, Imperial iGEM 2014 team has made it freely available in AddGene or upon request. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4541
  Illegal EcoRI site found at 1297
  Illegal SpeI site found at 1207
  Illegal PstI site found at 1685
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1297
  Illegal EcoRI site found at 4541
  Illegal SpeI site found at 1207
  Illegal PstI site found at 1685
  Illegal NotI site found at 1678
  Illegal NotI site found at 4547
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1297
  Illegal EcoRI site found at 4541
  Illegal XhoI site found at 1145
  Illegal XhoI site found at 1151
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4541
  Illegal EcoRI site found at 1297
  Illegal SpeI site found at 1207
  Illegal PstI site found at 1685
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4541
  Illegal EcoRI site found at 1297
  Illegal XbaI site found at 4556
  Illegal SpeI site found at 1207
  Illegal PstI site found at 1685
  Illegal NgoMIV site found at 2860
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1198
  Illegal BsaI.rc site found at 987