Composite

Part:BBa_K316027

Designed by: IC 2010 Team   Group: iGEM10_Imperial_College_London   (2010-10-26)


B. subtilis transformation vector with LacI, targets amyE locus.

This vector has been designed using the amyE 5' and 3' integration sequences for integration into B.subtilis genome


AmyE locus This vector has been designed using the amyE 5' BBa_K143008 and 3' BBa_K143009 integration sequences for integration into B. subtilis genome. Insertion into the amyE locus provides a selection marker as the bacterium will no longer be able to breakdown starch. An iodine assay can be used to confirm integration. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments.


LacI With constitutivly expressed LacI BBa_K143033 the vector is suitable for B. subtilis transformations with promoter hyper-spank BBa_K143015 or other LacI regulated gene-circuits.


Chloramphenicol Resistance This vector also contains a positive selection marker. Chloramphenicol acetyltransferase BBa_J31005 provides resistance to chloramphenicol antibiotic.


Blunt end cloning site PmeI restriction site BBa_K316013 is designed as a blunt ended DNA (eg. PCR product) insertion site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once. To add any BioBrick construct to the vector for integration, it may be linearised by PCR (or by cutting with NotI if further cloning steps are not required) and ligated into the vector after it is cut with PmeI.


VectorLacI.PNG

Figure I. Complete amyE vector for B. subtilis genome integration with LacI expression.

Warning: Check the notes in the experience page before proceeding to any wet lab work involving this part.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2922
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1141



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