Difference between revisions of "Part:BBa K1321300"

Revision as of 06:13, 2 November 2014

pSEVA331-Bb - pSEVA331 with biobrick prefix and suffix

This is pSEVA331 converted into a biobrick compatible format by substituting the original multiple cloning site for biobrick prefix and suffix. pSEVA331 is a broad host range plasmid, capable of replication in E.coli and several other organisms. It is capable of replication in the cellulose producing bacterium Gluconacetobacter xylinum. We have used it as our standard backbone for G.xylinus genetic engineering, in 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.. pSEVA331 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. 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