Team:Thessaly/Design
Project Design
Overview
The Amalthea project proposes a complete method of confronting IBDs, containing the diagnosis of IBDs, the evaluation of the gut microbiota, and the personalized treatment according to the gathered data. It also enables wireless and real-time evaluation of the microbiome, which is accomplished through the cooperation of the biological system and the electronic devices. Combining sensing, computation, and communication in a non-invasive manner, the capsule leverages the advantages of each approach, compared to currently used methods.
Aim
We aim to accomplish the communication between our inner world and the one outside of it, through the understanding of our body’s needs. This will be possible by identifying metabolite deficiencies directly correlated to IBD, exploiting a bio-electronic interface to enable real-time monitoring on the patient’s smartphone. Our final goal is the creation of a micro-bio-electronic ingestible device that will support physicians to provide help to their patients based on their identified needs. This will be used primarily by those who suffer from IBDs, contributing to the improvement of their well-being and quality of life.
Detection System
Diagnosis
The first step is the diagnosis of IBDs. This can be accomplished with a diagnostic tool consisting of engineered aptamers and toehold switches (Chau THT et al., 2020), that can detect salivary Calprotectin, providing a cell-free diagnostic test. When levels of Calprotectin are above normal, aptamers bind to their target, Calprotectin, and triggers the toehold switch activating the reporter gene. These measurements will be used to determine the stage of the inflammation. For the validation of the precision of the biomarkers for predicting and monitoring response to treatment, we will combine the results of the Calprotectin levels with the evaluation of the gut microbiota, by measuring the levels of SCFAs.
Evaluation of the gut microbiota
Recent events regarding the COVID-19 pandemic resulted in reviewing our goals and following a different path. This pathway led us to Phase I and the difficult choice to choose one aspect of our project. Due to this situation, we performed experiments that involved this part of our project and tackled the issues that came up (Read more).
Capsule & Bioelectronic system
The second step is the manufacturing of the capsule and its bioelectronic system, which is separated into two channels. In the first channel, the bacteria are contained, bordered from the electronics, that consist of the second channel. One critical parameter to take into consideration, when building an ingestible device, is the capsule size, so the proposed capsule’s dimensions are 21mm x 12 mm (length x diameter). The electronics are coated with a 1 mm thin Parylene-C membrane and then the whole device is cast into a PDMS capsule 12 mm in diameter, to be protected from the caustic gastrointestinal environment (Mimee et al., 2018). Concerning the power supply, a silver oxide button battery that provides the device 3V is equipped (Kourosh et al., 2017) (Figure 1.). This device meets the requirements on size and power, while also giving a good balance between range and human tissue penetration.
NOT-GATE
The device employs a semipermeable membrane to allow small molecules, such as SCFAs, to penetrate and react with the bacteria. This bacteria-based system possesses a NOT-GATE genetic circuit that performs a logical negation on its input, inverting the initial signal and representing the opposite logic-level to the input (Singh V., 2014). In the absence of SCFAs, the biosensor is activated so the reporter System is expressed, producing an electrochemical signal. Then the signal generated is processed and transmitted to a smartphone for convenient readout(Figure 2).
Figure 2. Image of a NOT-GATE circuit. The current output is inverted, therefore positive (1 or ON) becomes negative (0 or OFF), whilst negative (0 or OFF) would become positive (1 or ON).
Tango-GPCR Module
The diagnostic module has the form of a Tango-GPCR Module coupled to a downstream reporter system. More specifically, the GPCR receptor, that can be activated by endogenously present SCFAs, FFRA2, used for this module is fused with a repressor with a linker that contains a cleavage site, TCS, for a specific protease, TEV. Activation of the receptor recruits beta-arrestin fused with the protease that then cleaves and releases a repressor, LacI, to alleviate the expression of the reporter gene. The absence of SCFAs allows the expression and activation of the Reporter Module, allowing the physician to diagnose gut microbiota distortion (Barnea et al., 2008). The Reporter Module is an electrochemical module that transforms the biological signal into an easily detectable electrical one, enabling real-time monitoring (Vanarsdale et al., 2020). (Figure 3)
Tet-off Module & Prom Module
Τo provide a proof-of-concept (Read more), we tried to simulate the basic logic gate and organic structure of our Tango Module, by respectively designing a simple NOT- gate circuit based on the Tet-off Module and a simplified promoter system, the Prom Module.
The Tet-off module will simulate the circumstances of the logic gate behind the Tango Module regulating the activity of genes. This will be accomplished by the administration of tetracycline-derivatives that will silence the gene expression. In the absence of aTC, that is simulating the absence of SCFAs, the TetR is bound to the TRE and the transcription unit is blocked. These result in inhibition of LacI leading to a lac-regulated expression of the reporter gene. In the presence of aTC, TeTR is blocked, so LacI is expressed, leading to a lac- regulated inhibition of a reporter gene, eCFP (Das et al., 2016). (Figure 4)
The Prom Module, that we designed, can be considered a proof of concept since its structure is based on the NOT-GATE circuit. It consists of three basic parts, the promoters that are induced by SCFAs, a repressor protein, LacI, and a reporter gene, eCFP. The promoter pFliC (Tobe, Nakanishi and Sugimoto, 2010), that is more sensitive to butyrate and the promoter prpBCDE (Tsang, Horswill and Escalante-Semerena, 1998), that is more sensitive to acetate and propionate. The presence of SCFAs leads to the expression of LacI, concluding to the suppression of the signal. On the contrary, the lack of SCFAs obstructs the expression of LacI, thus allowing the activation of the reporter gene, eCFP. (Figure 5. & 6.)
Reporter Module
Our go-to Reporter Module is Tyrosine-Tyrosinase Electrochemical Gene Circuit that transforms the biological signal into an electrochemical one, enabling real-time monitoring. It is activated when tyrosine is produced, as a reporter gene.Then, due to the presence of tyrosinase, tyrosine is converted to L-DOPA and L-DOPA quinone, which can be detected and quantified electrochemically. That occurs through a redox reaction that results in the conversion of tyrosine to oxidative derivatives, a process that produces an electric current. The current can be detected with the use of electrodes attached below each channel. When it flows through the electrodes, the electrical signals generated are processed by the microcontroller and transmitted wirelessly to an external receiver (Vanarsdale et al., 2020). (Figure 7)
Therapeutic System
The therapeutic system has the form of a bacteria-based system capable of producing SCFAs. As mentioned before SCFAs can be used in multiple ways, including therapeutics, considering their anti-inflammatory properties and their effect on the gut microbiota. The microbiome plays a substantial role in controlling the normal function of the intestine and contributes to the deflection of inflammation and systemic disease (Ho SM, et. al., 2019). A crucial aspect of our project is to create personalized treatments for the patients by replenishing the missing amount of SCFAs, which will be provided by probiotics that will initiate their production (Markowiak-Kopeć et al., 2020).
Metabolic Engineering
Our goal is the production of SCFAs, in order to replenish the missing amount of them. To achieve that, we tap into common pathways of metabolism (e.g Glycolysis, Krebs Cycle) and engineered them.
For the production of acetate: We overexpress the genes of the C2-fermentive pathway that leads to the formation of acetate. When acetyl-CoA is formed we convert it to acetate by overexpressing 2 genes: Pta - Phosphate Acetyltransferase and AckA – Acetate Kinase. (Miscevic et al., 2018)
For the production of propionate : We tap into the Tricarboxylic Acid Cycle , and when the succinyl-CoA is formed , we convert it to propionate adding 3 genes from the Sleeping Beauty Operon. The Sleepign beauty mutase (Sbm) Operon , in E.coli , contains genes similar to those that take part in the oxidative propionate pathway of Propionobacteria. : Sbm – methylmalonyl-CoA mutase, YgfG: methylmalonyl- CoA decarboxylase and last YgfH: propionyl-CoA/succinyl- CoA transferase (Akawi et al., 2015)
For the production of butyrate: we tap into glycolysis and when acetyl-CoA is formed with the use of 6 genes we convert it to butyrate: BktB - β-ketothiolase , Hbd - (S)-3HB-CoA dehydrogenase, Crt - Crotonase, Ter - trans-2-enoyl-CoA reductase, Ptb- phosphate butyryltransferase , Buk - butyrate kinase. Some of the genes we add are from Butyrogenic Bacteria, such as Clostridium acetobutylicum and Treponema denticola. (Miscevic et al., 2018)
In order to reduce the carbon flux, we knockout genes associated with aerobic glycolysis, promoting the anaerobic glycolysis. Furthermore, to increase the yield, we express a membrane sugar transporter, “feeding” and enhancing the pathway of glycolysis. (Figure 8)
Compilation & transmission of bio-electronic information
In the second channel, our device utilizes the compilation and transmission of bio-electronic information from the gut. The biological signal is transformed into an electrochemical one by the tyrosine-tyrosinase system. That occurs through a redox reaction that results in the conversion of tyrosine to oxidative derivatives, a process that produces an electric current. The current can be detected with the use of electrodes attached below each channel. When it flows through the electrodes, the electrical signals generated are processed by the microcontroller and transmitted wirelessly to an external receiver. A simple RF transmission device operating in the 433 MHz commercial communication band - ideal for biomedical applications- is selected for the transmission (Caffrey et al., 2015) (Figure 9)
The bio-electronic information generated will be instantly stored in the cloud on the user’s smartphone or computer, for convenient readout. It will be accessible, at any given time, to patients and to professionals, who will use it to evaluate the microbiome and visualize the functionality of the intestinal flora. This information is then used to provide a personalized treatment based on the needs of each patient, to achieve relief of symptoms, and to design a daily diet without dietary restrictions.
GB- Assembly
We use GB assembly for the design of all the constructs above.
GoldenBraid (GB) is a DNA assembly strategy for Plant Synthetic Biology based on Type IIS
enzymes. It is also compatible with MoClo assembly. The sequences must not contain BsmBI
and BsaI sites! Domestication may be done in order to vanish BsaI and BsmBI sites from the
inner sequence.GB proposes a Modular Cloning System making fragments (promoters,
terminators, CDSs, etc) called ‘level 0’ subparts. Then combining the desirable fragments
from level 0; ‘level a’ cloning is succeeded by creating Transcription Units (TU). In the end,
desirable TUs are combined to result in ‘level Ω’ cloning. (Sarrion-Perdigones et al., 2013)
● Using BsmBI for Level 0 modules and ‘level omega’ (Level Ω)
● Using BsaI for ‘Level alpha’ (Level a)
But this is not the end, GoldenBraid outperforms Golden Gate, because of the ability to continuously clone TUs in an “exponential” manner, compared to the linear progression of Golden Gate. Binary assembly of 2 Level Ω (Level 2 for MoClo) results in an alpha vector. Again Binary assembly of 2 alpha results in an omega vector. With this assembly, you can insert step by step as many parts and as many TUs you want with high efficiency.
In a modular strategy, predefined categories, the so-called “parts,” are assembled together following a number of rules known as the “assembly standard.” Modular DNA building offers a number of advantages such as speed, versatility, laboratory autonomy, combinatorial potential, and often lower cost (Ellis et al., 2011).
This way the structures will be interchangeable and accessible to anyone who wishes to use the GB method.
Ιn addition, we have also created a GoldenBraid-based vector toolkit aiming to adjust the GoldenBraid assembly method to facilitate the heterologous expression of proteins in bacteria, because it was originally developed for Plant Synthetic Biology. For this purpose, we have designed proper customized SEVA-derived α & Ω GB vectors to allow gene cloning and expression in bacteria.
For our purpose, we have adjusted the GoldenBraid assembly strategy for Plant Synthetic Biology based on Type IIS enzyme, to facilitate heterologous expression in bacteria.
Safety
Due to various parameters, the interaction among the capsule, the genetically engineered bacteria, and human cells faces many safety, security, and ethical risks. The first barrier that protects us and the environment is the capsule itself that acts as a mechanical barrier, but we designed a “kill switch”, as a second barrier, in order to ensure that we can keep the patients safe. More information on our Safety Page (Read more).
References
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