Part:BBa_K823023

pSB_Bs1C: Empty backbone for integration into Bacillus subtilis amyE locus

This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the amyE locus and can be selected with Chloramphenicol (cat gene). It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1662 ([http://www.ncbi.nlm.nih.gov/pubmed/8973347 Guérout-Fleury et al.]).

This version still contains NgoMIV restriction sites, therefore is not RFC25-compatible. For an RFC25-compatible version please use the BGSC ECE257 or BBa_J179000.

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

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

The transformation into B. subtilis is explained here.

To check for integration of this vector into the amyE locus, the starch test can be used. The colonies obtained from the transformation into B.subtilis were streaked on an LB-Agar plate with 5 mg/L chloramphenicol and onto a starch plate (also W168 as a control) and incubated at 37°C over night. The starch plate is then covered with Lugol's iodine. Colonies without a bright surrounding are correct. We tested this vector with various inserts, for example the final construct of our Sporobeads with GFP. The starch test shows that the insertion in in the amyE locus and fluorescence microscopy reveals the functionality of the construct.

Also the iGEM-Team from [http://2012.igem.org/Team:Groningen/international_cooperation Groningen 2012] verified that this backbone works just fine.

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 6084
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 6090
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6084
  Illegal BamHI site found at 6066
  Illegal XhoI site found at 3574
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 6084
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 6084
  Plasmid lacks a suffix.
  Illegal XbaI site found at 6099
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 1128
  Illegal NgoMIV site found at 3538
  Illegal NgoMIV site found at 4677
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 4501