Part:BBa_K316022

B.subtilis transformation vector, targets amyE locus

AmyE locus This vector has been designed using the amyE 5' BBa_K143001 and 3' BBa_K143002 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.

Chloramphenicol Resistance This vector also contains a positive selection marker, flanked by two dif sites. Chloramphenicol acetyltransferase BBa_J31005 provides resistance to chloramphenicol antibiotic. Dif BBa_K316002 sites allow excision of the antibiotic marker after integration, thus allowing the same marker to be used again or as a precaution against horizontal gene transfer.

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.

Please see ‘Part Design’ section for design considerations and parts used.

Figure I. Complete amyE vector for B. subtilis genome integration.

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 529
  Illegal BglII site found at 1492
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]