PROJECT DESIGN
"The future is not to be forecast, but created."
- Arthur C. Clarke
"The future is not to be forecast, but created."
- Arthur C. Clarke
During the whole development of our project, we evaluated different organisms to find the most suitable for tackling aflatoxin B1 contamination. A first design was proposed for the model organism Escherichia coli, although later in the project, Saccharomyces cerevisiae was chosen as the most viable organism for the implementation of our solution and testing in the laboratory.
Therefore, we produced two whole different sets of parts, one for each chassis, and both intended to generate a genetic circuit that will allow our modified organism to perform the following new functions:
Sense AFB1
Adsorb and report AFB1 upon such recognition
To achieve this purpose, we introduce genetic elements exogenous to our organisms, which we organize into two discrete interacting modules:
Sensing module: coding for the machinery capable of binding AFB1 and transducing an intracellular signal in response.
Output module: activated by the signal derived from the sensing module and containing the information for effector and reporter molecules.
Following this general architecture we defined our genetic designs both for Saccharomyces cerevisiae and Escherichia coli, which are now described.
Living systems, from bacteria to multicellular organisms, use specialized receptors to detect environmental stimuli and trigger adaptive responses for survival. However, evolutionary constraints limit the range of inputs organisms can process.
Synthetic receptors offer a solution to expand their sensing capabilities. While many have been engineered by repurposing sensor domains from other species, antibody-derived ligand binding domains (LBDs) provide a more versatile tool for creating highly specific detection platforms.
Based on this premise, we engineered a transmembrane AFB1 synthetic receptor inspired in the GEMS platform (Scheller, et al., 2018), where activation is caused by ligand-induced dimerization of extracellular LBDs, which in turn brings together dimerization-dependent signal transduction domains in the intracellular side.
Our receptor is composed of two separate subunits which are succesfully targeted to the plasma membrane thanks to the attachment of the signal peptide from SUC2, a secreted invertase. There, it can come in contact with extracellular molecules including aflatoxin B1. Both receptors subunits exhibit a similar and highly modular structure with the following domains:
Schematic representation of S. cerevisiae receptor: Ligand binding domains (LBDs), consisting of two different anti-AFB1 scFvs, are highlighted in yellowish colours
Two different scFvs (single-chain variable fragments) drive receptor heterodimerization upon aflatoxin B1 exposure, thanks to their simultaneous binding to the toxin on distinct epitopes.
Both scFvs, namely scFv1 and scFv2, were taken from the work developed by the previous iGEM team Tsinghua 2017, since they had been shown to promote dimerization of fused domains upon binding of AFB1.
The LBDs are also linked by a short flexible peptide (SGEF) to the scaffold anchored to the membrane to allow movement and favour interaction between the paratope and the toxin (Scheller, et al., 2018).
Both scFvs consist of fusion proteins made of the variable regions of the heavy and light chains of highly avid anti-AFB1 immunoglobulins, both connected by a peptide linker of ~20 amino acids, rich in serine (S) and glycine (G) for flexibility, and other charged amino acids for solubility (Chen et al., 2013).
Schematic representation of S. cerevisiae receptor: The EpoR scaffold, central to GEMS and composed of a transmembrane (TM) and extracellular (D2) domain, is highlighted in purple
This region provides membrane anchorage and promotes transmission of the signal from the extracellular to the intracellular side. It consists of the transmembrane and extracellular D2 domain from the erythropoietin receptor (EpoR) (P118-S272).
This scaffold represents the core element of the GEMS platform and has been shown to restrict ligand-independent dimerization. It has been repeatedly used in mammalian systems and has also been successfully implemented in yeast.
Schematic representation of S. cerevisiae receptor: Intracellular signalling relies on the split-ubiquitin-mediated release of a synthetic transcription factor, shown in green. C-terminal and N-terminal fragments of ubiquitin (Cub and NubG) are marked in dark blue and light blue, respectively
The signaling capacity of the receptor is provided by an intracellular platform based on the split ubiquitin system (Snider et al., 2010), which is capable of mediating the release of an attached transcription factor.
N-terminal and C-terminal halves of ubiquitin are attached to each of the receptor subunits. These fragments, called Nub and Cub, comprise the residues M1-I35 and G36-G77 of the native protein, respectively. Both Nub and Cub independently can not be recognized by cellular deubiquitinases, and spontaneous reconstitution of the split protein is prevented through mutation I13G, rendering NubG.
Only when NubG and Cub are brought into close proximity upon AFB1 exposure and receptor dimerization, can the ubiquitin be reconstituted and recognized by cellular deubiquitinases, leading to hydrolysis and release of the transcription factor fused to the C-terminal end of Cub.
The protein released is a synthetic transcription factor (sTF) designed to provide orthogonal gene expression under specific synthetic promoters, avoiding cross-talk with other cellular transcriptional machinery. It is formed by the fusion of LexA DNA binding domain and Herpesvirus VP16 activation domain. Thanks to this structure, it can exert potent transcriptional activation on DNA sequences harboring LexA operators followed by native core promoter elements (Rantasalo et al., 2016).
The output module permits a maintained and effective cellular response against aflatoxin B1. The key regulatory element is a promoter formed by four LexA operators and the core promoter from Saccharomyces cerevisiae TDH3 gene (~200 bp), which is activated by the sensing module.
Upon receptor dimerization, the transcription factor released binds to the mentioned promoter and activates the transcription of three components involved in the cellular response:
A highly-expressed VHH provides detoxification capacity to our organism. We utilize the anti-AFB1 Nb28 S102D VHH (He et al., 2021) and accesibility to the antigen is maximized by fusion with Aga2 and its signal peptide, which contribute to sorting of the protein and its anchor to the cell surface by binding the transmembrane protein Aga1 (Wang et al., 2005).
To keep a strong response once the first stimulus has been received, the transcription factor that initially triggered the expression through cleavage from the receptor, is synthesized in its free form, allowing for a positive feedback that ensures sustainability over time.
Finally, to enable detection and genetic construction verification, an easily identifiable protein is produced. Our own construction harbors the coding sequence for yEmRFP (yeast enhanced monomeric RFP) as a first approach due to its visually spottable nature.
High levels of synthesis of these three proteins is achieved by using a single regulated promoter thanks to the addition of the IGG6 DNA sequence.
IGG6 is a 9-bp motif that resulted from the optimization of a fungal intergenic sequence, IGG1, known to generate bicistronic expression in Glarea lozoyensis. The resulting IGG6 has been proven to permit polycistronic expression in other fungal species and is thought to act by mediating translation re-initiation by the ribosome, producing separate proteins from each of the coding sequences of a transcript (Yue et al., 2023). This element allows us to support the production of our three proteins while avoiding exccesive complexity of the design.
Overview of IGG6-mediated polycistronic expression: the IGG6 sequence promotes translation re-initiation by the ribosome, permitting the separate translation of consecutive CDSs in an mRNA
Altogether, the genetic circuit implemented is able to give an effective response to aflatoxin B1 exposure, following a simple logic:
Animated overview of Saccharomyces cerevisiae's circuit behaviour
Apart from the genetic constructs designed to function in the final version of our organism, we generated three modules aimed to test the functionality of each part of our circuit independently.
As a first approach to demonstrate the AFB1-dependent dimerization capacity of scFv1 and scFv2 in the context of a transmembrane receptor based on the GEMS platform, we applied the technique of bimolecular fluorescence complementation (BiFC).
We designed 2 receptor subunits harboring the scFvs and EpoR scaffold, but with a split mCerulean as intracellular domain. Assessment of the fluorescence intensity of this receptor in response to aflatoxin B1 will provide information on the capacity of reconstitution of split proteins upon AFB1-induced dimerization with the receptor architecture proposed.
Split-mCerulean receptors expressing device; SP: signal peptide, mCer: monomeric Cerulean
To evaluate the detoxification potential of our yeast, the same surface VHH intented for expression upon receptor activation was introduced in a constitutively expressed unit harboring the strong TDH3 promoter.
Transcriptional unit constitutively expressing the anti-AFB1 VHH, expressed at surface by fusion with Aga2 and its signal peptide
Finally, to check the functionality of IGG6, the key component of our output module, we constructed a polycistronic gene containing the coding sequences for three fluorescent proteins: yeast-enhanced monomeric RFP (yEmRFP), yeast-enhanced GFP (yeGFP) and yeast enhanced CFP (yECFP), all of them expressed under the same TEF1 promoter.
Polycistronic construction containing three IGG6-separated CDSs, coding for the fluorescen proteins yEmRFP, yeGFP and yECFP
As mentioned, despite not being the final organism utilized for assays in the laboratory, we still designed a sensing-response platform in the model organism Escherichia coli, whose viability is supported by a wide literature.
In this case, we constructed a synthetic receptor built upon the EMeRALD system, which is based on the engineering of E. coli’s CadC protein. CadC is a transmembrane transcriptional activator from the ToxR family which harbors cytosolic split-DNA binding domains (split-DBDs) which can be activated by receptor dimerization.
CadC counts with a periplasmic pH-sensitive domain and is also responsive to lysine concentration due to interaction of its transmembrane region with the lysine permease (LysP). Substitution of these regions with desired alternative domains renders a receptor unresponsive to its natural regulators and with specificity to a novel ligand, in our case: AFB1 (Chang et al., 2018).
Schematic representation of E. coli receptor: Ligand binding domains (LBDs), consisting of two different anti-AFB1 scFvs, are highlighted in yellowish colours
Similarly to our Saccharomyces cerevisiae receptor, heterodimerization of two distinct subunits is required for the functionality of the platform. This is enabled thanks to the same scFv1 and scFv2 (Tsinghua, 2017), with different codon optimizations.
Both scFvs are linked to the rest of the receptor through a GS-rich flexible linker which facilitates movement and distancing from the membrane (Chen et al., 2013).
Schematic representation of E. coli receptor: transmembrane segment of the receptor, formed by an aritificial Leu(16) helix, is indicated with a dark purple line
An artificial transmembrane helix composed of 16 leucine residues, Leu(16), is used to span across the membrane and eliminate lysine-dependent regulation. This artificial helix permits transmission of the signal upon LBDs dimerization while restricting spontaneous dimerization of unbound receptors. It also favours membrane insertion due to its highly hydrophobic nature (Lindner et al., 2014).
Schematic representation of E. coli receptor: Signaling depends on the dimerization of the split-DBD from CadC protein, highlited in blue
Both receptor subunits harbor the same intracellular domain, consisting of the cytosolic split-DBD from the native CadC one-component system.These split-DNA binding domains are not functional in their monomeric form. However, upon dimerization, transcriptional activation is made possible under the CadBA promoter (Kuper et al., 2005).
After AFB1 exposure and signaling through the sensing module, expression of genes downstream of the CadBA promoter is triggered, deploying the following two molecular responses:
Detection is made possible thanks to the synthesis of a visually identifiable product, which was chosen to be mRFP for our proof of concept.
The AFB1 adsorption capacity of the bacterium is augmented upon toxin exposure due to the induced expression of the receptor subunits themselves. These are synthesized under their own regulated promoter and provide a strong binding affinity through their LBDs, at the same time that they keep transducing a signal, establishing a positive feedback which maximizes performance of the system.
Overall, both modules acting together generate a circuit which allows aflatoxin B1 sensing and a rapid response to the toxin with the following expected behavior:
Animated overview of Escherichia coli's circuit behaviour
As it has been exposed, both our genetic designs exhibit a highly modular nature. This valuable property allows for re-engineering of the constructions through exchange of ligand binding domains or effector molecules.
This poses a valuable strategy to tackle toxins or molecules different from AFB1, as well as to deploy novel or more complex responses upon stimulus. We propose to facilitate this adaptability through the implementation of an extended MoClo for the construction of GEMS and EMeRALD-based receptors, as described in our Contribution page.
Therefore, we propose two alternative designs to incorporate a new AFB1-sensitive pathway that allows detection and adsorption of the toxin, both in Saccharomyces cerevisiae and Escherichia coli. From both of the functions achieved, arise several applications to combat the hazard posed by this mycotoxin.
With Saccharomyces cerevisiae being the most applicable to treat aflatoxin B1 contamination, as describen in our Engineering, both of the designs offer a versatile architecture which can be repurposed for coping with other toxins or molecules of interest.
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