Plasmid_Backbone

Part:BBa_K799025

Designed by: Daniel Wolozny   Group: iGEM12_Johns_Hopkins_Wetware   (2012-09-25)

pSB1C3 yGG ORF acceptor vector

The original pSB1C3 backbone has been adapted so that yeast Golden Gate (yGG) ORF (open reading frame) parts, conforming to RFC88, can be quickly converted into BioBrick parts that are compatible with the BioBrick assembly standard.

Between the BioBrick prefix and suffix we have introduced two recognition sequences for the type IIS restriction enzyme BsaI. The BsaI sites flank an RFP gene and are oriented to eliminate themselves and the RFP during digestion and ligation of a yGG ORF part. The 4 base pair sticky ends generated by BsaI digestion of the modified pSB1C3 vector are complementary to the 4 base pair overhangs in our Yeast Golden Gate ORF parts, thus enabling yGG to BioBrick promoter part conversion.

The best way to use this part is to perform a simultaneous digestion-ligation reaction, whereby the yGG promoter construct, the modified pSB1C3 vector, BsaI, and T4 DNA ligase are all introduced into a single tube and incubated at 30C for 1 hour, 50C for 5 minutes, and 80C for 5 minutes. Following E.coli transformation, selection on chloramphenicol plates will eliminate the background of cells transformed with the original yGG promoter construct entirely. Newly assembled constructs, in which the yGG ORF part has been converted into a BioBrick part, will make white colonies compared to the red colonies produced by intact, RFP-containing pSB1C3 vector.

Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal XhoI site found at 1012
    Illegal XhoI site found at 1904
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Plasmid Map

Psb1c3 orfAV.png

Sequencing

OrfAV sequencing.png

The vector was sequenced from the prefix, across its RFP biobrick and till the suffix. This was done to prove that the prefix and suffix remained the same after ligation of a part through the Golden Gate method.

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