Composite

Part:BBa_K4190019

Designed by: Zoe L. Petroff, Neo Peng   Group: iGEM22_UCSC   (2022-09-22)

Exendin-4 Gene Insert for S. cerevisiae

His-tagged Exendin-4 coding region to be inserted into a plasmid via Golden Gate Assembly and expressed in E. coli.

Restriction Enzyme Sites

For both E. coli and S. cerevisiae, we used the type IIS restriction enzyme, BsaI, on our insert. This restriction enzyme binds to the sequence GGTCTC and cuts after the first and fifth non-specific nucleotides, which results in “sticky ends” downstream of the binding site [1]. The restriction enzyme site was present on the top and bottom strands respectively in the five prime to three prime direction. The desired insert sequence was constructed to allow scarless integration into pET:28-GFP.

Histidine Tag

After the start codon for our desired gene code, the first element is the histidine tag (his-tag). Our his-tag is made up of six histidine amino acids. When translated, the his-tag will be connected to the N-terminus of our Exendin-4 (Ex-4) protein. This allows us to verify that our desired protein was expressed through protein purification [2].

Amino Acid Linker and Protease Cut Site

We used a ten amino acid linker of alternating glycine and serines to distance our his-tag from the Ex-4 polypeptide, and enhance his-tag exposure for protein purification. Furthermore, we include a four amino acid enterokinase protease cut site. This allows us to use enterokinase to cleave the his-tag from our protein once it is expressed. Because of the way Ex-4 binds to the GLP-1 receptor [3], we do not need to cut the histidine tag off of our protein after it is purified.

Codon Optimized Protein Sequence

The amino acid sequence for Ex-4 was found on the Genscript website [4]. This same website was used to optimize the codon sequence for E. coli, and the S. cerevisiae sequence was optimized through IDT [5]. The optimized codon sequence will produce our functional protein when transcribed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 6
    Illegal BsaI.rc site found at 358


References

[1] J. H. Lee, H. J. Won, E.-S. Oh, M.-H. Oh, and J. H. Jung, “Golden Gate Cloning- Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants,” Front. Plant Sci., vol. 11, p. 559365, Oct. 2020, doi: 10.3389/fpls.2020.559365.

[2] “His-Tag Purification | Chromatography Media, Columns and Kits | Bio- Rad.” https://www.bio-rad.com/featured/en/his-tag-purification.html (accessed Aug. 27, 2022).

[3] “GLP1R - Glucagon-like peptide 1 receptor - Homo sapiens (Human) | UniProtKB | UniProt.” https://www.uniprot.org/uniprotkb/P43220/entry (accessed Aug. 27, 2022).

[4] GenScript.” GenScript. https://www.genscript.com/ (accessed July 20, 2022).

[5] “Codon Optimization Tool | IDT,” Integrated DNA Technologies. https://www.idtdna.com/pages/tools/codon-optimization-tool (accessed Aug. 27, 2022).

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