Part:BBa_K316027

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 hyper-spank BBa_K143015 controlled gene-circuits.

Chloramphenicol Resistance This vector also contains a positive selection marker, flanked by two dif sites. Chloramphenicol acetyltransferase BBa_J31005 provides resistance to chloramphenicol antibiotic.

Blunt end cloning site PmeI restriction site BBa_K316013 is designed as a cloning site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once.

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

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
  

For more information about our project please visit our wiki [http://2010.igem.org/Team:Imperial_College_London] or take the tour [http://2010.igem.org/Team:Imperial_College_London/Tour/Page_One] to learn more about the project.