Part:BBa_K316020

B. subtilis genome integration vector, targets pyrD locus

PyrD This vector has been designed using the Pyrd 5' BBa_K143001 and 3 BBa_K143002' integration sequences for integration into B. subtilis genome. Insertion into the pyrD locus provides a negative selection marker as the bacterium will no longer be able to synthesise uracil. Thus medium supplement of 40ug/ml is required for growth. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments.

Spectinomycin Resistance This vector also contains a positive selection marker, flanked by two dif sites. Aad9 BBa_K143065 provides resistance to spectinomycin antibiotic. Dif sites BBa_K316002 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 pyrD 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 475
  Illegal BglII site found at 1550
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 208
  Illegal SapI.rc site found at 1123
  

For more information about our project please visit our wiki [http://2010.igem.org/Team:Imperial_College_London].