Part:BBa_K1321302

pSEVA331-BB with J23100-RFP

This part contains Anderson promoter J23117 and RFP coding device in pSEVA331-BB backbone. pSEVA331-BB is a broad host range plasmid converted into biobrick format, and is capable of replication in E. coli and several other Gram- bacterial species. We have verified that it also replicates in the cellulose producing bacterium Komagataeibacter rhaeticus. BBa_K1321302 is a member of the Komagataeibacter genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332). BBa_J23100 drives a high level of expression in K. rhaeticus.

Figure 1. RFP expression from BBa_K1321302 in K. rhaeticus iGEM strain. Top three plates - transformed K. rhaeticus, bottom - untransformed control. Image taken under blue light box 5 days after transformation. K. rhaeticus was cultured on HS-agar plates, at 30degC inverted.

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, with quality control provided (see Experience).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3776
  Illegal SpeI site found at 37
  Illegal PstI site found at 920
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3776
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 37
  Illegal PstI site found at 920
  Illegal NotI site found at 913
  Illegal NotI site found at 3782
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3776
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3776
  Illegal SpeI site found at 37
  Illegal PstI site found at 920
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3776
  Illegal XbaI site found at 3791
  Illegal SpeI site found at 37
  Illegal PstI site found at 920
  Illegal NgoMIV site found at 2095
  Illegal AgeI site found at 616
  Illegal AgeI site found at 728
  
• 1000
  COMPATIBLE WITH RFC[1000]