Part:BBa_K5466022

Constitutive expression of receptor antiAFB1-scFv2 Cub

The expression of receptor antiAFB1-scFv2 Cub (BBa_K5466019) is controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure it is always available for the detection of AFB1.

Device the express a component of the signaling platform Patrol Yeast. The Cub component of the split-ubiquitin system with scFv2 to detect AFB1 in yeast. Use with constitutive expression of receptor antiAFB1-scFv1 Nub (BBa_K5466021).

Dimerization of Cub and NubG occurs only when both scFvs bind to aflatoxin, this reconstitutes the ubiquitin, leading to hydrolysis and release of the transcription factor, activating the expression of (BBa_K5466011).

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 2412
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Patrol Yeast

In the split-ubiquitin system for detecting protein interactions, ubiquitin is expressed as two distinct fragments in separate plasmids: the N-terminal fragment, NubG, and the C-terminal fragment, Cub. A the transcription factor is linked to the C-terminus of Cub, and when NubG is co-expressed, the two fragments of ubiquitin reassemble, forming a split-ubiquitin heterodimer that leads to the release of the transcription factor. This system have been repurposed for application as synthetic receptors by substituting the “bait” and “prey” proteins with scFvs or sdAbs, which can promote ligand-induced dimerization of fused domains, restoring their ability to activate gene expression and producing a response to the target.

One of this approaches, named Patrol Yeast, drew inspiration from GEMS on the use of EpoR as a receptor scaffold to create a signaling platform in S. cerevisiae based on the split-ubiqutin system. Since the transmembrane (TM) signaling capability of sensor proteins is crucial for detecting molecules outside of cells, Su et al., (2022) utilized the EpoR D2 and TM regions in Yeast Patrol to link the LBD and split ubiquitin within the cells. This resulted in a system composed of LBD-EpoR-NubG and LBD-EpoR-Cub-TF proteins which was demonstrated to admit scFvs and sdAbs (including VL and VH chains) as ligand binding domains

To complete the Yeast Patrol signaling platform to detect AFB1 you need to express this part with antiAFB1-scFv2 Cub (BBa_K5466021).

LexA-VP16 (BBa_K5466010) was used due to being a orthogonal synthetic transcription factor, to avoid unpredictable interference with the functionality of synthetic systems. Additionally, its potent activation domain ensures strong expression of the desired response upon AFB1 detection. In our project it is a triple response (BBa_K5466020).

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

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

Scheller, L., Strittmatter, T., Fuchs, D., Bojar, D., & Fussenegger, M. (2018). Generalized extracellular molecule sensor platform for programming cellular behavior. Nature Chemical Biology, 14(7), 723-729. https://doi.org/10.1038/s41589-018-0046-z

Su, J., Zhu, B., Inoue, A., Oyama, H., Morita, I., Dong, J., Yasuda, T., Sugita-Konishi, Y., Kitaguchi, T., Kobayashi, N., Miyake, S., & Ueda, H. (2022). The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning. Biosensors And Bioelectronics, 219, 114793. https://doi.org/10.1016/j.bios.2022.114793