Composite

Part:BBa_K3114023:Design

Designed by: Cassandra Sillner, Sara Far, Sravya Kakumanu, Nimaya De Silva, Andrew Symes   Group: iGEM19_Calgary   (2019-10-08)


6xHis-tagged modified water-soluble chlorophyll binding protein (ModGIX) circuit


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 654
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 179
    Illegal AgeI site found at 122
    Illegal AgeI site found at 438
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 765


Design Notes

When designing this circuit and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, the basic parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part was constructed using component parts listed below. It is iGEM Type IIS RFC[1000] compatible and BioBrick RFC[10] compatible.

The circuit was designed for inducible, high-level expression and has been codon optimized for E. coli. A 6X Histidine affinity chromatography tag was added to the N-terminus of the ModGIX coding sequence for purification.

Source

This part was synthesized.

References

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765