Part:BBa_K1321307

pSEVA351-Bb

pSEVA351-Bb is pSEVA351 converted into a biobrick-compatible format by substitution of the native MCS with biobrick prefix and sufix. It is a broad host-range plasmid, capable of replication in E.coli as well as the cellulose-producing bacterium Gluconacetobacter xylinus (see Figure 1) and other Gram- bacteria. It can be used for G.xylinus genetic engineering and is a member of the G.xylinus toolkit (parts BBa_K1321295 - BBa_K1321332).

Figure 1. Confirmation of pSEVA321, pSEVA331, pSEVA351, pBla-Vhb-122 and pBAV1K ability to replicate in G.xylinus.. pSEVA351 can replicate in G.xylinus. G.xylinus igem strain was transformed with pSEVA321-Bb using electroporation. Transformed cells were plated out on HS-Cam plates. Single colonies were then picked and grown in 5ml HS-cellulose medium (in 50ml Corning tubes) at 180rpm, 30C for 3 days. Cultures were then miniprepped using Qiagen DNA mini kit (see protocol here)". Miniprepped DNA was then used as template for pSEVA-specific primers. PCR products were visualized using 1% agarose gel, 100V, 20min. Ladder - NEB 2logL, expected band size for positive control - 328bp; size of lowest bright band of the ladder-500bp.

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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. pSEVA351-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
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5024
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 5030
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5024
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 5024
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 5024
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5039
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1191
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.