Part:BBa_K5466036

Constitutive expression of AntiAFB1-scFv1 EpoR Split-N-mCerulean

The expression of surface AntiAFB1-scFv1 EpoR Split-N-mCerulean to observe if, in the presence of aflatoxin, dimerization occurs and mCerulean is reconstituted. Use with constitutive expression of AntiAFB1-scFv2 EpoR Split-C-mCerulean (BBa_K5466031).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2034
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

mCerulean

mCerulean is cyan fluorescent protein that can be use in vitro and in living cells, engineered from ECFP.

mCerulean originated from ECFP, which, despite several spectroscopic disadvantages such as a low quantum yield, low extinction coefficient, and a fluorescence lifetime best described by a double exponential, was commonly used. To improve ECFP's properties for FRET measurements, a site-directed mutagenesis approach was employed by Rizzo et al. (2004) to address these limitations. The resulting variant, named Cerulean by Rizzo et al. (2004) (ECFP/S72A/Y145A/H148D), exhibited a significantly improved quantum yield, higher extinction coefficient, and a fluorescence lifetime best fit by a single exponential. Cerulean is 2.5 times brighter than ECFP.

BiFC

Studying protein–protein interactions (PPIs) is essential for understanding the molecular mechanisms that drive protein function. These interactions can vary between cells, occurring in different locations and with varying strengths. To fully grasp protein function, it’s crucial to capture PPIs in their natural cellular context and conditions. Bimolecular fluorescence complementation (BiFC) facilitates the study of PPIs in diverse native contexts and has proven effective in various cell types, from bacteria to mammals. This technique relies on the ability of monomeric fluorescent proteins to reassemble from two separate fragments when in close proximity. When candidate proteins are fused to these complementary fragments, a fluorescent signal is generated upon interaction, enabling the visualization of weak and transient PPIs. BiFC can also be used to investigate multiple PPIs simultaneously using a multicolor approach.

In addition to detecting PPIs, BiFC provides insights into the subcellular localization and affinity of these interactions in living cells. However, BiFC is irreversible, which, unlike FRET, prevents the analysis of dynamic complex formation and dissociation. Nonetheless, BiFC enhances the detection of weak PPIs by stabilizing them, even at normal expression levels. It can also be applied to investigate distinct PPIs simultaneously using a multicolor setup. This is achieved by complementing N-mVenus with C-mCerulean, which has a different excitation and emission wavelength than mVenus and mCerulean.

Since the Patrol Yeast signaling platform also uses a system originally developed to study PPIs and is based on split-ubiquitin, we thought that BiFC was an ideal technique, due to its similar mechanisms, to reliably confirm that the LBDs intended for use in the signaling platform produced ligand-induced dimerization.

scFv

A single-chain variable fragment (scFv) is a fusion protein made up of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, connected by a flexible peptide linker of 10 to 25 amino acids. This linker enhances solubility and maintains the structural integrity necessary for antigen binding, allowing scFvs to retain the specificity of the original antibody despite lacking constant regions.

scFvs offer several advantages over full-length monoclonal antibodies, including reduced side effects due to the absence of the Fragment crystallizable (Fc) region, simpler construction and expression, and improved pharmacokinetic properties. Each scFv contains two variable domains with three hypervariable complementary determining regions (CDRs) responsible for binding to antigens, with varying contributions to specificity. For instance, the CDR3 of the heavy chain significantly contributes to binding specificity, while CDR2L has a minor role. The stability of scFvs is crucial for their effective use in both in vitro and in vivo applications.

This scFv and (BBa_K5466014) were selected because, in the iGEM17_Tsinghua project, from which these parts originate, they are used in an intracellular biosensor based on the Yeast Two Hybrid system, meaning that interaction occurs in the presence of aflatoxin. We chose them because since they produced a signal in that system, we expected that they could produce ligand induced dimerization of the receptors.

References

Jia, Y., Bleicher, F., Merabet, S., & Reboulet, J. (2021). Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein–Protein Interactions in Human Living Cells. Methods In Molecular Biology, 173-190. https://doi.org/10.1007/978-1-0716-1593-5_12

Muñoz-López, P., Ribas-Aparicio, R. M., Becerra-Báez, E. I., Fraga-Pérez, K., Flores-Martínez, L. F., Mateos-Chávez, A. A., & Luria-Pérez, R. (2022). Single-Chain Fragment Variable: Recent progress in cancer diagnosis and therapy. Cancers, 14(17), 4206. https://doi.org/10.3390/cancers14174206

Rizzo, M. A., Springer, G. H., Granada, B., & Piston, D. W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnology, 22(4), 445–449. https://doi.org/10.1038/nbt945

Weber-Boyvat, M., Li, S., Skarp, K., Olkkonen, V. M., Yan, D., & Jäntti, J. (2014). Bimolecular Fluorescence Complementation (BiFC) Technique in Yeast Saccharomyces cerevisiae and Mammalian Cells. Methods In Molecular Biology, 277-288. https://doi.org/10.1007/978-1-4939-2309-0_20