Part:BBa_K5466023

Constitutive expression of AFB1 detection platform

The expression of signaling platform Patrol Yeast to detect AFB1.

IGG6/NAL10 (BBa_K5466001) allow co-transcription of antiAFB1-scFv1 NubG (BBa_K5466018) and antiAFB1-scFv2 Cub (BBa_K5466019) reducing the number of base pairs required for the expression of both receptors. By utilizing IGG6, the total construct size is reduced by approximately 1000 bp compared to the combined 5558 bp of (BBa_K5466021) and (BBa_K5466022) required for the same function. This optimize the size of the genetic construct. The expression is controlled by the constitutive promoter TDH3 (BBa_K124002) to ensure it is always available for the detection of AFB1.

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 3825
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

IGG6/NAL10

To reduce the length and complexity of genetic constructs, systems for polycistronic expression are required, permitting the expression of several genes from a single promoter and avoiding a multiple promoter strategy.

Yeast Patrol

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-scFv1 NubG (BBa_K5466018).

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, in this particular scFv, of 15 amino acid. 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.

These 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

Yue, Q., Meng, J., Qiu, Y., Yin, M., Zhang, L., Zhou, W., An, Z., Liu, Z., Yuan, Q., Sun, W., Li, C., Zhao, H., Molnár, I., Xu, Y., & Shi, S. (2023). A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nature Communications, 14(1). https://doi.org/10.1038/s41467-023-40027-0