Team:Thessaly/Engineering
Engineering
Overview
To tackle malnutrition and IBDs, we need a robust and comprehensive design to account both for project and experimental challenges. Thus, we have used the engineering principles of Abstraction Hierarchy and Modularity, and have split our project into two systems, the Detection and Therapeutic System. Within each system, there are several modules and devices that help us demonstrate the functionality of the project in an iterative and realistic manner. Explore our engineering design below.
-Modular Cloning/Expression Vectors:
Amalthea is composed of many systems, modules and devices, which translates to a lot of transcriptional units (TUs) that need to be cloned in appropriate vectors in the lab. We created six cloning/expression vectors, combining a bacterial-expression variation of the GoldenBraid standard (Sarrion-Perdigones et al. 2013), with the help of SEVA plasmids.
We designed a GoldenBraid-based vector toolkit aiming to adjust the GoldenBraid DNA assembly method for heterologous expression of proteins in bacteria, since it was originally developed for plants. For this purpose, we created proper customized
α (alpha)& Ω (omega) GB vectors, using the SEVA plasmid collection, to allow modular cloning and expression in bacteria.
Go to results: Click here!
pDGB1 alpha 1R (α1R): BBa_K3505008
pDGB1 alpha 2 (α2): BBa_K3505009
pDGB1 omega 1R (Ω1R): BBa_K3505010
pDGB1 omega 2 (Ω2): BBa_K3505011
The SEVA collection is a plasmid repository for complex prokaryotic phenotypes. The collection entails most antibiotic resistance genes and ORIs, which can be used in any combination using rate restriction enzyme sites.This way, deconstruction and reconstruction of a plasmid and repurposing (e.g. transfer of a 5 TU construct from a cloning vector to an expression vector) is simple. (Martínez-García et al. 2020).
We used SEVA43 and SEVA23 as backbones to create the alpha and omega vectors. The alpha vectors have Kanamycin Resistance and the Omega vectors have Spectinomycin Resistance, to follow the GoldenBraid standard. pBBR, a low/medium copy ORI, was chosen to facilitate heterologous protein expression. However, due to low yield of the plasmid, downstream cloning was hindered and difficulty was increased. In our next Design-Build-Test cycle, We will use the modularity of the SEVA backbones to replace the low copy ORI by a high copy (pRO1600/ColE1).
Using our system, the whole SEVA collection can be utilized to create vectors with a desirable of antibiotic resistance genes and ORIs by targeting the T0 and T1 terminators that are universal.
Go to Parts overview: Click here!
“Detection System”
Overview: The purpose of the Detection System is to provide real-time monitoring of intestinal health, using Short-Chain Fatty Acids (SCFAs) as a biomarker.
-GPCR-Tango Module:
To detect clinically relevant concentrations of SCFAs, we designed a synthetic GPCR coupled with a NOT-GATE device. By combining a bacterial version of the Tango Assay (Dogra, Sona, Kumar and Yadav, 2016) and an inversion of signal output, we managed to detect the absence of SCFAs.
To achieve clinically relevant detection of SCFAs (butyrate, propionate, acetate) absence with one component, we designed a GPCR-mediated NOT-GATE module, which combines a typical NOT-GATE device with a bacterial version of the Tango Assay , which is mainly used in eukaryotes. The FFAR2 GPCR (Kaemmerer, 2010) is shown to bind SCFAs and facilitate downstream signalling in vivo
This module consists of four transcription units:
1. an inducible promoter for arabinose (ParaBAD):GPCR FFAR2: a V2 tail: a cleavage site-TCS:LacI: double terminator
2. an inducible promoter for arabinose (ParaBAD):b-arrestin:TEV protease, terminator
3. an constitutive promoter (AndersonJ23115):lac operator and reporter gene - (CFP):double terminator
4. a constitutive promoter (AndersonJ23115) :RraA :double terminator
The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death. (Kallunki, Barisic, Jäättelä and Liu, 2019).
Figure 1 : GPCR -Tango Module
The existence of V2 tail permits the recruitment of a b-arrestin , that is combined with a TEV protease that recognizes and cleaves the cleavage site. When TEV protease cleaves , the lac repressor is released and binds to the lac operator ,inhibiting the transcription of reporter gene. In the presence of SCFAs the GPCR is activated. Membrane proteins(MP) are pharmaceuticals targets and have a crucial role in cellular functions. However , MP overexpression result in cytotoxicity and cell death. In order to prevent that , we decide to co-express rraA , resulting in a dual positive effect on recombinant GPCR expression :
1. the expression of rraA , suppresses the cytotoxicity , thus resulting in enhanced levels of expression and bacterial biomass
2. contribute to cellular accumulation and result in better-folding of a MP (prokaryotic and eukaryotic origin)
(Gialama et al., 2017)
This module , due to an error in the orientation of omega1R , isn’t complete. This construct isn’t the final one. The final construct was placed on an alpha1 vector which would consist of 2 omegas: omega1 and omega2. The construction of omega 1, was wrong, so we created omega1R. We didn’t have enough time to repeat vector construction experiments this year. Our purpose is to assemble and test the functionality of the GPCR-Tango Module, once we have access to the lab and report our results in iGEM 2021. See you in Paris!
-Tet-off module
To reduce the complexity and liabilities of bacterial GPCR expression, we designed a simple anhydrotetracycline (aTC)-induced NOT-GATE device, that has the same logical structure as the GPCR-Tango module, but uses the well-characterized transcriptional factor Tetracycline Repressor (TetR) (Gossen and Bujard, 1993).
This iGEM year was unique in many aspects. Because of that, we didn’t test all the devices/modules that we designed and built. However, we managed to do some initial characterization on our lower complexity devices. We designed and built a simple TetR-based on the NOT-GATE to prove the functionality of the device in our hands, before moving on to a SCFA-induced module.
In the presence of the strong chemical repressor anhydrotetracycline (aTC), TetR inhibition is blocked and the transcription of LacI is allowed. LacI in turn binds to Lac Operator (LacO) operator and inhibits the expression of a reporter gene.
In absence of aTC, TetR binds to the tetracycline response element-TRE and inhibits LacI expression. This inhibition allows the expression of the reporter gene. To obtain quantifiable and visual output, we used eCFP as a reporter gene.
1. constitutive promoter (AndersonJ23115) : Tet repressor : double terminator
BBa_K3505033
2. constitutive promoter (AndersonJ23115) : Tet operator : lac repressor : double terminator
BBa_K3505034
3. constitutive promoter (AndersonJ23115) :lac operator : ECFP: double terminator
BBa_K3505032
4. stuffer
Figure 2 : Tet-off module
Our initial design included eGFP as the reporter gene. When we completed the cloning experiments, we immediately started plate-reader assays before sequencing results arrived. No detectable fluorescence was produced in our initial experiments testing the construct LacO-eGFP.
After sequencing results came in, we noticed some consistent single-point mutations in our positive eGFP clones, which when BLASTed returned eCFP as the best alignment. It turns out that, due to wrong starting template, we had cloned a eCFP instead of eGFP, which was the reason for the non-detectable fluorescence. After adapting the plate-reader parameters to fit eCFP excitation/emission spectra, fluorescence was produced from the bacteria, expressing the construct.
This module, due to an error in the orientation of omega1R , isn’t complete. This construct isn’t the final one. The final construct was placed on an alpha1 vector which would consist of 2 omegas: omega1 and omega2. The construction of omega 1, was wrong, so we created omega1R. We didn’t have enough time to repeat vector construction experiments this year. Our purpose is to assemble and test the functionality of the Tet-off Module, once we have access to the lab and report our results in iGEM 2021.
To our results: Click here
Prom Module
To provide a failsafe in case the GCPR-Tango Module is not functional, we designed a promoter-based module for SCFAs detection. We used SCFA-induced promoters, pFliC (Tobe, Nakanishi and Sugimoto, 2010) and prpBCDE (Tsang, Horswill and Escalante-Semerena, 1998) to allow SCFAs to act as an input to the NOT-GATE device.
The next step in our design, was a NOT-GATE device that is activated by SCFAs. To do that, we integrated a set of SCFA-inducible promoters to regulate the expression of LacI. In the presence of the SCFAs, LacI is activated and binds to Lac Operator (LacO) operator, inhibiting the expression of a downstream reporter gene.
In absence of SCFAs, LacI inhibition is blocked and transcription of the reporter gene takes place. To obtain quantifiable and visual output, we used eCFP as a reporter gene to obtain comparable data with our Tet-Off Module.
1. inducible promoter (pFliC) :: lac repressor : double terminator
BBa_K3505027
2. constitutive promoter (AndersonJ23115) :lac operator: ECFP: double terminator
BBa_K3505032
Figure 4 :Prom module. pFliC induced from Butyrate
Figure 5 :Prom module prpBCDE induced from Acetate and Propionate.
To our proof of concept: Click Here
-Tyrosine-Tyrosinase Reporter Module
To obtain an output that can be easily digitized, we integrated a Tyrosine-Tyrosinase Reporter Module to couple SCFAs detection with an electrochemical signal. This can be achieved by the SCFA-induced overexpression of key Tyrosine metabolism enzymes.
For our Reporter Module, we made use of the ability of L-Dopa to produce electrochemical signal using a recently reported Tyrosine-Tyrosinase module. The electrochemical signal was chosen because of its high quality, easier digitalization and more accurate transmission to an external user (VanArsdale et al., 2020).
Adhesin Involved in Diffuse Adherence (AIDA) consists of three parts, Signal Peptide, Passenger and the transmembrane region. (Hörnström, Larsson, van Maris and Gustavsson, 2019)
Figure 6 : Tyrosinase converting L-Tyrosine to L-Dopa and L-Dopa quinone.
The signal peptide is cleaved, in order for the protein to be translocated to the membrane, while the Tyrosinase, that we add, acts as a Passenger. We chose the Tyr1 gene coding Tyrosinase because it is the smallest of all Tyrosinases available and lacks cysteines, resulting into an easier transportation to the outer membrane. (Gustavsson et al., 2016)
We used a constitutive promoter for the regulation of the Tyr1-AIDA. Having in mind the concept that Tyrosinase must be there waiting the L-Tyrosine to appear. This led to reduced cell growth and reduced expression of the protein, which we hypothesized is caused by toxicity. In the next Design-Build-Test cycle, we will add an inducible promoter and express antitoxic proteins (Gialama et al., 2017) for better expression.
To our results: Click here
“Therapeutic System”
The purpose of the therapeutic system is to provide the essential elements that IBDs patients lack and relieve their symptoms – SCFAs.
SCFAs production: We integrate metabolic pathways for three key SCFAs – butyrate, propionate, acetate – in E. coli to overproduce each one separately. The production from the bacteria will take place either as probiotics producing in vivo the SCFAs, or ex vivo and then oral consumption as a prebiotic.
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).above-heading
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 re- ductase , 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)
Figure 7 : Production of SCFAs taking advantage of common metabolic Pathways.
Optimization of SCFAs production: To optimize SCFAs production, knockouts of key enzymes are needed to redirect the carbon flux appropriately. We will design knockout experiments using CRISPR/Cas9, ssDNA recombineering or a combination of the two to obtain strains optimized for SCFAs production. Stay tuned!
“Dry Lab”
For the evaluation of the patient’s microbiome, we decided on a non-invasive approach that will bring no pain or discomfort to the patient, while also presenting effective and safe transmission of the data to the experts. Thus, we agreed upon using an ingestible capsule, which is rendered safe and effective by applying the following materials in its manufacturing process.
The capsule and its bioelectronic system is separated in two channels. The first channel contains the detection bacteria with the semipermeable membrane. We selected an ABS plastic (Protolabs Inc.) Semipermeable membrane (0.22μm pore size, EIMF22205, Millipore Sigma) for the purposes of our project (Mimee et al., 2018).
The bacterial channel is connected with the electronic channel via the detection electrodes. We selected a CHI101 ⌀2mm gold working electrode, a CHI115 platinum counter electrode and a CHI 111 Ag/AgCl reference electrode from CH Instruments. Those specific electrodes were picked according to papers that support their effectiveness at tyrosine/tyrosinase reactions (VanArsdale et al., 2020).
A custom PCB (Advanced Circuits) was selected as a board for the electronics, while a basic PIC18F Microcontroller is employed for the calculations and control of the capsule’s function. The microcontroller works well with our capsule requirements, as it is light, has a low power consumption and works effectively in the temperature of the human body.
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 re- ductase , 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)
Two Duracell Silver Oxide 364 batteries with supply voltage of 1.5 V each, electric charge of ~80mAh and a maximum current of 70mA were picked as a power source for the capsule.
4-15μm of Parylene-C (Daisan Kasei Co.) was selected as coating for the electronics and also as a moisture barrier (Mimee et. al, 2018).
As for the antenna, it should be comprised of materials with high relative permittivity, medium conductivity, and preferably biocompatible. For a biocompatible encapsulation we have chosen Al2O3 (Degussit AL23, εr =9.9, tanδ = 0.0001) as practically lossless, biocompatible, and widely available commercially in various shapes and sizes (Nikolayev et al., 2017) .
For the substrate, we picked FR-4(εr = 4.8, tanδ = 0.026), as it is the most prevalent material in all the antenna designs that we studied and also very flexible to help roll the antenna easier within the capsule.
The ground plane and patch are made of annealed copper, due to its high electric conductivity and low cost.
The antenna modeling took place within the AntennaDesigner toolkit of MATLAB.
References
Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.
Gialama, D., Delivoria, D., Michou, M., Giannakopoulou, A. and Skretas, G., 2017. Functional Requirements for DjlA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR. Journal of Molecular Biology, 429(12), pp.1800-1816.
Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), pp.4411-4412.
Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J. and Larsson, G., 2016. Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior. Scientific Reports, 6(1).
Hörnström, D., Larsson, G., van Maris, A. and Gustavsson, M., 2019. Molecular optimization of autotransporter-based tyrosinase surface display. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1861(2), pp.486-494.
Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.
Kallunki, Barisic, Jäättelä and Liu, 2019. How to Choose the Right Inducible Gene Expression System for Mammalian Studies?. Cells, 8(8), p.796.
Mimee, M., Nadeau, P., Hayward, A., Carim, S., Flanagan, S., Jerger, L., Collins, J., McDonnell, S., Swartwout, R., Citorik, R., Bulović, V., Langer, R., Traverso, G., Chandrakasan, A. and Lu, T., 2018. An ingestible bacterial-electronic system to monitor gastrointestinal health. Science, 360(6391), pp.915-918.
Nikolayev, D., Zhadobov, M., Le Coq, L., Karban, P. and Sauleau, R., 2017. Robust Ultraminiature Capsule Antenna for Ingestible and Implantable Applications. IEEE Transactions on Antennas and Propagation, 65(11), pp.6107-6119.
Tobe, T., Nakanishi, N. and Sugimoto, N., 2010. Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli. Infection and Immunity, 79(3), pp.1016-1024.
Tsang, A., Horswill, A. and Escalante-Semerena, J., 1998. Studies of Regulation of Expression of the Propionate (prpBCDE) Operon Provide Insights into How Salmonella typhimurium LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways. Journal of Bacteriology, 180(24), pp.6511-6518.
VanArsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G., van Maris, A. and Bentley, W., 2020. A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks. ACS Synthetic Biology, 9(5), pp.1117-1128.
