Composite

Part:BBa_K3767003:Design

Designed by: Cecilia Hernandez-Hosaka & Griffin Watson-Boehnisch   Group: iGEM21_Queens_Canada   (2021-06-05)

3-24 ScFv sequence linked to Green Fluorescent Protein (GFP)

The 3-24 ScFv sequence is composed of light chains (VL) and heavy chains (VH) from antibodies that are linked by a peptide linker[1]. The sequence was made to detect the bacteria that result in Lyme Disease, known as Borrelia Burgdorferi using the OspA. The design of the 3-24 ScFv sequence was based on past research done by Ghosh and Huber to optimize its binding affinity to both the OspA and E. coli expression[2]. The green fluorescent protein (GFP) is a reporter gene that is derived from the jellyfish Aequeora victoria. It can be used as a marker for gene expression and protein targeting in intact cells due to its extremely visible fluorescence[3]. This part is being registered as a composite of a previously registered sequence (BBa_K3767000) and GFP (BBa_E0040).

Usage and Biology/Background

The green fluorescent protein is a reporter gene that consists of 238 amino acids and has a three amino acid sequence (Ser65-Tyr66-Gly67). It can form a structure to emit a visible green fluorescence when exposed to blue UV light[4]. As it is a marker protein, it can use its fluorescence to mark proteins to help allow specific proteins to be identified easily. The structure consists of a beta-sheet polypeptide, an alpha helix polypeptide, an alpha helix, which overall forms a beta barrel shape. The 3-24 ScFv sequence attaches to the C terminus. A chromophore in the centre is required to emit the fluorescence, which is done through the process of cyclization, dehydration, and oxidation.

The three amino acid structure undergoes these steps to produce a structure that contains the fluorescence emitting feature. This has an absorbance wavelength of 397 nm and goes up to 475 nm through deprotonation. A mechanism of the fluorescence process is shown below.


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.rc site found at 1379



References


1. Ahmad, Z. A., Yeap, S. K., Ali, A. M., Ho, W. Y., Alitheen, N. B., and Hamid, M. (2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology. 2012, 1–15

2. Ghosh, S., and Huber, B. T. (2007) Clonal diversification in OspA-specific antibodies from peripheral circulation of a chronic Lyme arthritis patient. Journal of Immunological Methods. 321, 121–134

3. Tsien, R. Y. (1998) The Green Fluorescent Protein. Annual Review of Biochemistry. 67, 509–544

4. Kauk, M. (2018) Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists