Part:BBa_K1321301

pSEVA321-Bb

This is pSEVA321 converted into a biobrick compatible format by substituting the original multiple cloning site for biobrick prefix and suffix. pSEVA321 is a broad host range plasmid, capable of replication in E.coli and several other bacterial species. We have verified that it also capable of replication in the cellulose producing bacterium Komagataeibacter rhaeticus and Gluconacetobacter xylinus (Figure 1). We have used it as a shuttle vector for Komagataeibacter genetic engineering. BBa_K1321301 is a member of the Komagataeibacter genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332).

Figure 1. Confirmation of pSEVA321, pSEVA331, pSEVA351, pBla-Vhb-122 and pBAV1K ability to replicate in G.xylinus.. pSEVA321 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.

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
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3572
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3578
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3572
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3572
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3572
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3587
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1191
  Illegal NgoMIV site found at 2076
  Illegal NgoMIV site found at 3187
  Illegal NgoMIV site found at 3311
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.