Designed by: Michael Florea   Group: iGEM14_Imperial   (2014-10-08)


pBAV1K-T5-gfp is a broad host range plasmid engineered by Bryksin&Matsumura (2010) based on the pWV01 RCR origin of replication. It has been shown to replicate in Escherichia coli, Acinetobacter baylyi ADP1, Agrobacterium tumefaciens, (all Gram-negative), Streptococcus pneumoniae, Leifsonia shinshuensis, Peanibacillus sp. S18-36 and Bacillus subtilis. Imperial iGEM2014 team also showed that it can replicate in the cellulose producing bacterium Gluconacetobacter xylinus. This plasmid contains a gfp gene behind T5 promoter and a KanR selectable marker. pBAV1K-T5-gfp can not be requested from the registry due to binding materials-transfer agreements, however it can be bought from Addgene

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

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.'

References: Bryksin AV, Matsumura I (2010) Rational Design of a Plasmid Origin That Replicates Efficiently in Both Gram-Positive and Gram-Negative Bacteria. PLoS ONE 5(10): e13244. doi:10.1371/journal.pone.0013244 Sequence and Features

Assembly Compatibility:
  • 10
    Illegal suffix found in sequence at 810
    Illegal XbaI site found at 15
  • 12
    Illegal SpeI site found at 811
    Illegal PstI site found at 825
    Illegal NotI site found at 6
    Illegal NotI site found at 818
  • 21
    Illegal BamHI site found at 609
    Illegal XhoI site found at 510
  • 23
    Illegal suffix found in sequence at 811
    Illegal XbaI site found at 15
  • 25
    Illegal XbaI site found at 15
    Illegal SpeI site found at 811
    Illegal PstI site found at 825
  • 1000