Part:BBa_K1321332

promoter(lacI regulated)-RFP in pSEVA331-Bb

This is RFP expressed behind a lacI regulated promoter (part BBa_R0010) in pSEVA331-Bb plasmid backbone (part BBa_K1321300). This construct is a member of the G.xylinus genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332). Expression from lacI regulated promoter is constitutive in G.xylinus even in the absence of lactose, indicating that LacI is not present in the cell. This notion is supported by the fact that blastn or tblastx of lacI coding region against existing G.xylinus genomes does not result in any matches even at low-stringency settings.

Figure 1. RFP expression from BBa_K1321332 in G.xylinus igem strain. Top three plates - transformed G.xylinus, bottom - untransformed control. Image taken under blue light box 5 days after transformation. G.xylinus was cultured on HS-agar plates, at 30degC inverted.

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 3949
  Illegal suffix found in sequence at 1078
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3949
  Illegal SpeI site found at 1079
  Illegal PstI site found at 1093
  Illegal NotI site found at 1086
  Illegal NotI site found at 3955
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3949
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3949
  Illegal suffix found in sequence at 1079
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 3949
  Illegal XbaI site found at 3964
  Illegal SpeI site found at 1079
  Illegal PstI site found at 1093
  Illegal NgoMIV site found at 2268
  Illegal AgeI site found at 781
  Illegal AgeI site found at 893
  
• 1000
  COMPATIBLE WITH RFC[1000]