Part:BBa_K1185004

Integration vector for Bacillus subtilis derived from pSac-Cm

This plasmid was derived from pSac-Cm by insertion of biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS). New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies. This backbone has a multi host replication origin and replicates in E. coli. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences, and in order to check for integration a simple Phenol Red Sucrose test can be perform, if sacA is no longer functional, they will remain ph7, if they are still able to digest sucrose their pH will be under 5.

Testing and Characterisation

Groningen team 2012 have showed that this plasmid is functional in E. coli. This plasmid can be used as cloning tool in E. coli by selecting the white colonies from the red colonies after transformation (the plasmid in the red colonies has no insert; the RFP sequence is not replaced by the insert).

This plasmid backbone is designed to be used as an integration plasmid in B. subtilis, but they did not show that the backbone is actually functional.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 1
  Illegal suffix found at 5190
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  Illegal SpeI site found at 5191
  Illegal PstI site found at 5205
  Illegal NotI site found at 7
  Illegal NotI site found at 5198
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  Illegal BamHI site found at 163
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 1
  Illegal suffix found at 5191
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 1
  Plasmid lacks a suffix.
  Illegal XbaI site found at 16
  Illegal SpeI site found at 5191
  Illegal PstI site found at 5205
  Illegal NgoMIV site found at 4107
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 2034
  Illegal SapI.rc site found at 3116