Plasmid

Part:BBa_K1033206:Design

Designed by: Anders Edlund, Viktor Blomkvist, Mikael Strandgren, Jens Isaksson   Group: iGEM13_Uppsala   (2013-09-25)
Revision as of 22:29, 3 October 2013 by MikaelS (Talk | contribs) (→‎Source)

Lactobacillus shuttle vector pSBLbC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 3036
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3036
    Illegal NheI site found at 1981
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3042
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3036
    Illegal BamHI site found at 1960
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 3036
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 3036
    Illegal XbaI site found at 3051
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The basic design is an iGEM backbone where we have changed the replicon and resistance cassette through subcloning and mutagenesis PCR.

The starting backbone was pSB4C15 made by Erik Lundin at Uppsala University by modifying pSB1C3. It is modular, meaning it was made with unique restriction sites around all key parts and we simply had to sublcone. We amplified the broad range replicon from pJP059 with the corresponding restriction sites (MluI and NheI), and did the same with the backbone to remove the old replicon, then cut and ligated them.

We kept the resistance gene, but had to change it's promotor since we suspected it wouldn't work well in Lactobacillus, this we did with PCR amplicitation with the new promotor in the overhangs.

The overhang is a standard RFP (BBa_J04450), but it may have to be replaced to use for screening in Lactobacillus.

The replicon and resistance cassette are surrounded by restriction sites selected by Erik Lundin for their usefullness, non-complementarity and not conflicting with BioBrick standards (excepting 12). The replicon uses MluI and NheI, and the resistance cassette uses SacI and SalI.

Source

The backbone comes from pSB4C15 which is a modification of pSB1C3.

The replicon comes from the plasmid pJP059.

The new promotor in the resistance cassette (CP29: BBa_K1033222) comes from the promotor library we synthesized to BioBrick standard based on the article "The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters." by Peter Ruhdal Jensen and Karin Hammer, shown below.[[[1]]]

References

The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters PETER RUHDAL JENSEN AND KARIN HAMMER Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 21 October 1997 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/pdf/am000082.pdf