Part:BBa_K1065201

Pxyl+GFP in an integrative Bacillus subtilis specific backbone

This construct is the composition of the Bacillus subtilis specific integrative backbone (BBa_K823024)[1] and BBa_E0840(strong RBS BBa_B0030 + GFP BBa_E0040[2] + terminators BBa_B0010 and BBa_B0012). The backbone hosts the xylose inducible promoter pXyl (BBa_K143014) that controls the expression of the GFP gene.
Before the actual transformation this kind of construct has to be digested with the Sca1 restriction enzyme.
The backbone integrates in the thrC locus and confers spectinomycin resistance in Bacillus subtilis and ampicillin resistance in Escherichia coli.The ampicillin resistance gene hosts the restriction site for Sca1.

Usage and Biology

This construct was successfully transformed in Bacillus subtilis grown in a minimal medium from Groeningen iGEM team .
N.B. The pH of the medium needs to be around 7 and 8!
The integration in the thrC locus could be demonstrated through a threonine test because the transformed strain should become auxotroph for this aminoacid.
N.B. The pXyl promoter is strongly inhibited by glucose,so the medium for the induction has to be minimal!
In literature is reported the usage of the Spizizen minimal medium.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4773
  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4773
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4779
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4773
  Illegal BamHI site found at 4757
  Illegal XhoI site found at 1011
  Illegal XhoI site found at 1929
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4773
  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4773
  Illegal XbaI site found at 4788
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1893
  Illegal NgoMIV site found at 3032
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2856
  Illegal BsaI.rc site found at 5467
  Illegal SapI.rc site found at 605