Part:BBa_K3114021:Design

6xHis-tagged water-soluble chlorophyll binding protein (6GIX) circuit with TorA signal peptide

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 761
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 286
  Illegal AgeI site found at 229
  Illegal AgeI site found at 545
  
• 1000
  COMPATIBLE WITH RFC[1000]

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

This part was constructed using Golden Gate assembly and parts listed below. It is iGEM Type IIS RFC[1000] compatible because the BsaI sites are removed during assembly.

• T7 Promoter and strong RBS (BBa_K3114012)
• TorA signal peptide (BBa_K3114005)
• 6xHis-tagged 6GIX (BBa_K3114006)
• Double terminator (BBa_K3114013)

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 6GIX coding sequence for purification.

Source

This part was generated using Golden Gate assembly using various parts that were 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