Plasmid_Backbone

Part:BBa_J179000

Designed by: Jara Radeck   Group: Mascher_lab   (2013-12-03)

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 (Guérout-Fleury et al.).

This version is derived from BBa_K823023 and does not contain AgeI nor NgoMIV restriction sites, therefore is RFC25-compatible.

This part was also evaluated in the publication The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis by Radeck et al..


This BioBrick is part of the 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.



LMU-Munich-Starchplate.png
280px-LMU Firstspore.jpg

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.








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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 4501
    Illegal EcoRI site found at 6084
    Illegal XbaI site found at 6099
    Illegal SpeI site found at 2
    Illegal PstI site found at 16


[edit]
Categories
//chassis/multihost
//chassis/prokaryote/bsubtilis
//chassis/prokaryote/ecoli
//plasmid/chromosomalintegration
//plasmidbackbone/assembly
//plasmidbackbone/operation
Parameters
chassisB. subtilis + E. coli
copies
origin
resistanceA(E. coli) C(B. subtilis)