Part:BBa_K1321299

J23101-GFP in pSEVA321-Bb

This is sfGFP expressed behind a LacI regulated promoter (part BBa_R0010) in pSEVA321-Bb plasmid backbone (part BBa_K1321301). This construct is a member of the G.xylinus genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332). BBa_K1321299 was confirmed via PCR and sequencing. Transformation of BBa_K1321299 into E.coli DH10B results in a low level of sfGFP expression even without addition of lactose after 3 days of incubation at 37degC on LB-chloramphenicol plates. BBa_K1321299 is currently being characterized in G.xylinus.

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

NOTE: Because the registry's standard plasmid backbone pSB1C3 is not capable of replication in Gluconacetobacter species, the G.xylinus 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 G.xylinus toolkit available for the synthetic biology community, Imperial iGEM 2014 team has made it freely available upon request, with quality control provided (see Experience). To request, please contact Imperial iGEM 2014 team.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4499
  Illegal suffix found in sequence at 928
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4499
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 929
  Illegal PstI site found at 943
  Illegal NotI site found at 936
  Illegal NotI site found at 4505
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4499
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4499
  Illegal suffix found in sequence at 929
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4499
  Illegal XbaI site found at 4514
  Illegal SpeI site found at 929
  Illegal PstI site found at 943
  Illegal NgoMIV site found at 2118
  Illegal NgoMIV site found at 3003
  Illegal NgoMIV site found at 4114
  Illegal NgoMIV site found at 4238
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 706