Generator

Part:BBa_K2319000:Design

Designed by: Raj Magesh Gauthaman   Group: iGEM17_IISc-Bangalore   (2017-09-25)


sfGFP-SpyCatcher under T7 expression system


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1200
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 768
    Illegal XhoI site found at 1001
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 42
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 57


Design Notes

HindIII, ATG, AgeI

A HindIII restriction site, a start codon and an AgeI restriction site are added immediately downstream of the T7 promoter+RBS. A number of design considerations motivated the choice of these restriction sites. The HindIII site (A\AGCTT) — sandwiched between the RBS and the start codon — has a sequence very similar to the optimal sequence predicted by the sequence logo of E. coli ribosome binding sites, which can be found here. In fact, the HindIII sequence is closer to the optimal sequence than the typical 5'-TACTAG-3' mixed SpeI-XbaI restriction site formed by BioBrick assembly, improving the initial ribosome-mRNA binding and improves translation.

The AgeI site was chosen to simplify assembly of fusion proteins in this backbone: by inserting a protein coding sequence at the N-terminus of the existing protein using the HindIII and AgeI sites, a fusion protein can be formed with a benign scar. The AgeI site (A\CCGGT) is translated in-frame to Thr-Gly, amino acids commonly used in linker sequences for fusion proteins. Threonine's hydroxyl group makes it hydrophilic — allowing stabilizing interactions with the aqueous cellular environment — while glycine's small size makes the linker more flexible, allowing both protein domains to fold independently. In addition, the AgeI site is useful if the user wishes to transfer an RFC25-compatible fusion protein (Freiburg format) into our expression system.

Linker

Serine, like threonine, has a hydroxyl group that contributes to stability by interacting with the aqueous cellular environment. Glycine, again due to its small size, maintains flexibility of the linker. This linker region also contains a BamHI restriction site (G\GATCC) whose in-frame translation is Gly-Ser. This BamHI site is used to link our two protein domains during assembly of the fusion construct.

Source

This part was generated by assembling the T7 promoter, a strong RBS, the sfGFP-SpyCatcher coding sequence, and a T7 terminator. All these parts are available in the Registry.

References