Plasmid_Backbone

Part:BBa_J179002

Designed by: Jara Radeck   Group: Mascher_lab   (2013-12-20)

pBs4S: Bacillus subtilis vector, thrC integration, spec resistance

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 is derived from BBa_K823022 and contains neither NgoMIV nor AgeI 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..

LMU-Munich-PSBBs4S.png


This BioBrick is part of the LMU 2012 igem project BacillusBioBrickBox

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

The transformation into B. subtilis is explained here.

LMU-Munich-Thrplate.png

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: 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2856
    Illegal EcoRI site found at 4552
    Illegal XbaI site found at 4567
    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/operation
Parameters
chassisB. subtilis + E. coli
copiesintegrative in B. subtilis
originpDG1731
resistanceA(E. coli) S(B. subtilis)