Part:BBa_K5466027

Autoregulated antiAFB1-scFv2 CadC receptor

The promoter leakage maintains low expression of the receptor. Since it is regulated by the promoter which is activated by the receptors, this allows for an increase in expression upon detection of aflatoxin, creating a positive feedback loop (until all AFB1 is captured), thereby saturating the receptor membrane and facilitating the capture of AFB1.

Use with Autoregulated antiAFB1-scFv1 CadC receptor (BBa_K5466026)

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
  COMPATIBLE WITH RFC[1000]

Positive autoregulation

Due to the inherent leakiness of bacterial inducible promoters, the receptor subunits are expressed at low levels under the control of the pCadBA (BBa_K3425101), producing a sufficient basal amount of receptors to detect AFB1 in the environment. Upon the presence of the toxin, receptor heterodimerization occurs, facilitated by scFvs on the extracellular side of the membrane. This leads to the reconstitution of CadC’s DNA-binding domain, allowing it to bind to the promoter and activate transcription. Consequently, more receptor proteins are expressed, increasing the surface density of anti-AFB1 paratopes and boosting the number of activated receptors, which in turn promote the synthesis of additional effector and reporter molecules.

CadC

CadC is a one component system (OCS). OCS consist of single proteins containing both sensor and output domains. The most studied system is Escherichia coli’s CadC transmembrane transcriptional activator, on which modification relies the technology of EMeRALD (Engineered Modularized Receptor Activated via Ligand-induced Dimerization).

CadC is a bitopic transmembrane protein with a cytoplasmic split-DNA binding domain and a periplasmic pH-sensor in which dimerization of the sensor domain triggers dimerization of the cytoplasmic DBD and transcriptional activation.

EMeRALD

EMeRALD exploits the modular structure of this one-component system to generate new receptors by swapping sensor domains and introducing desired ligand binding domains (LBDs). This approach has been proven compatible with LBDs from other one-component systems as well as antibody-derived domains including VHH and scFvs, boosting the range of ligands of application.

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_K5466015) 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.

Reference

Chang, H., Mayonove, P., Zavala, A., De Visch, A., Minard, P., Cohen-Gonsaud, M., & Bonnet, J. (2017). A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria. ACS Synthetic Biology, 7(1), 166-175. https://doi.org/10.1021/acssynbio.7b00266

Chang, H., Zúñiga, A., Conejero, I., Voyvodic, P. L., Gracy, J., Fajardo-Ruiz, E., Cohen-Gonsaud, M., Cambray, G., Pageaux, G., Meszaros, M., Meunier, L., & Bonnet, J. (2021). Programmable receptors enable bacterial biosensors to detect pathological biomarkers in clinical samples. Nature Communications, 12(1). https://doi.org/10.1038/s41467-021-25538-y

Ulrich, L. E., Koonin, E. V., & Zhulin, I. B. (2004). One-component systems dominate signal transduction in prokaryotes. Trends In Microbiology, 13(2), 52-56. https://doi.org/10.1016/j.tim.2004.12.006