Part:BBa_K823022

pSB_Bs4S: Empty backbone for integration into Bacillus subtilis thrC locus

This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1731.

This version still contains NgoMIV restriction sites, therefore is not RFC25-compatible. For an RFC25-compatible version please use the BGSC ECE259 or BBa_J179002.

This BioBrick is part of the LMU 2012 igem project [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks BacillusBioBrickBox]

For handling of B. subtilis vectors, please see here.

The transformation into B. subtilis is explained here.

We tested the integration of this plasmid with the threonine test: The colonies obtained from the transformation are streaked on three plates: LB with 100mg/L spectinomycine, minimal medium with threonine and minimal medium without threonine. As minimal medium, the MNGE-medium described in the transformation protocol was used. The plates are incubated at 37°C over night. Positive colonies grow on LB and minimal medium with threonine, but not on minimal medium without threonine.

Reference: [http://www.ncbi.nlm.nih.gov/pubmed/8973347 Guérout-Fleury et al.]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4552
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4558
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4552
  Illegal BamHI site found at 4534
  Illegal XhoI site found at 1011
  Illegal XhoI site found at 1929
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4552
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4552
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4567
  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]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2856
  Illegal SapI.rc site found at 605