Team:Thessaly/Engineering

Engineering

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Overview

Engineering represents a strategy of problem solving or development of new technologies and products. Under this perspective, assumptions and approximations have to be expressed through a thorough design in order to define the system's variables, possible outputs and behaviour under different conditions. In this page, we show how we performed our system’s modeling, concluded in our final hardware for the capsule and designed our wet lab experiments concerning both the capsule and the probiotic based on the DBTL cycle.

GPCR MODULE

PFLIC MODULE

TYROSINE-TYROSINASE MODULE

SCFAs PRODUCTION

CUMATE KILL-SWITCH

EXTRACELLULAR IPTG STABILITY

TYROSINASE AS LAC OPERON'S OUTPUT

HARDWARE

Tango GPCR metabolite detection module

Figure 1: The NOT GATE based TANGO GPCR SCFAs detection module, inhibiting the Signal in presence of SCFAs.

The TANGO GPCR SCFAs detection module is a whole-cell biosensor, hosted inside the tank inside the bioelectronic capsule beneath the Semipermeable membrane, that allows SCFAs to pass through. SCFAs activate the GPCR FFAR2  that is fused to a LacI  through a linker, containing a TEV Cleavage Site (TCS). After activation, Beta arrestin B-arr2 fused to a TEV protease cleaves and releases LacI, suppressing the reporter module. The aforementioned genes encode for eukaryotic transmembrane proteins that are toxic to bacteria, due to aggregate formation. To overcome this obstacle, we implemented a genetic circuit that favors bacterial expression of eukaryotic proteins (Michou, Stergios & Skretas, 2020). The final construct(BBa_K3866014) consists of four Transcription Units (TUs). The three of them are regulated by araBAD   BBa_K3505025 , BBa_K3505026 , BBa_K3505032 , BBa_K3505038 . We had placed the four TUs having reverse orientation in order to avoid Polymerase slippage using two alpha vectors: α2 BBa_K3505009 and α1 Reverse BBa_K3505008. We changed the TU's orientation to forward (Dusek et al., 2020), solving the problem of DNA loop formation because of the repeated araBAD sequences.

References

1. Dusek, J., Plchova, H., Cerovska, N., Poborilova, Z., Navratil, O., & Kratochvilova, K. et al. (2020). Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors. Frontiers In Plant Science, 11. doi: 10.3389/fpls.2020.522059


2. Michou, M., Stergios, A., & Skretas, G. (2020). SuptoxD2.0: A second‐generation engineered Escherichia coli strain achieving further enhanced levels of recombinant membrane protein production. Biotechnology And Bioengineering, 117(8), 2434-2445. doi: 10.1002/bit.27378

Promoter Flic Metabolite detection module

Figure 1: The NOT GATE based pFliC SCFAs detection module, inhibiting the Signal in presence of SCFAs.

The detection system of the capsule is based on the NOT-GATE device. The signal is in low concentrations of SCFAs. The promoter Flic is sensitive for SCFAs especially for butyrate (Tobe, Nakanishi and Sugimoto, 2010). We conducted experiments in order to verify the activation of pFlic by butyrate and the functionality of our module Level omega:LacO:ECFP:term-pflic:LacI:term (BBa_K3866036). During our experiments, we changed the fluorescent protein ECFP (BBa_K3866028) to sfGFP (BBa_K3866030), a stronger fluorescent protein, in order to be sure that the signal is not false positive and that the range between negative and positive control is distinct.

References

1. 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.

Tyrosine / Tyrosinase Reporter module

Figure 1: The arabinose inducible reporter module, Converting L-Tyrosine to L-Dopa and L-Dopa quinone for electrochemical output.

Figure 2: The metabolic pathway of L-Tyrosine conversion catalyzed by Tyrosinase for optical output.

Tyrosinase Tyr1 is an enzyme responsible for the conversion of L-Tyrosine to L-DOPA. Tyrosinase is transported to the extracellular membrane by the fused AIDA autotransporter and its signal peptide (BBa_K3866010), and the catalysis that performs, leads to the subsequent release of electrons while L-DOPA converts to L-DOPA quinone (VanArsdale, 2020). This electrical current can be detected and serve as the step of conversion from the biological to the electrochemical signal in our bioelectronic capsule. The first iteration we performed was choosing an arabinose inducible promoter araBAD (BBa_K3866000) for the production of our protein. This helped us overcome the metabolic strain of constitutive expression (BBa_K3505012). In order to make sure that our system is both induced and sensitive, we also worked with a range of different concentrations (0,002%, 0,02%, 0,2%, 1%, 2%, 6%, 12%) of the inducer molecule, L-arabinose.

References

1. VanArsdale, E., Hornstrom, D., Sjoberg, G., Jarbur, I., Pitzer, J., Payne, G. F., … Bentley, W. E. (2020). A co-culture based tyrosine-tyrosinase electrochemical gene circuit for connecting cellular communication with electronic networks. ACS Synthetic Biology. doi:10.1021/acssynbio.9b00469

SCFAs Production modules

Our proposed solution is a probiotic able to produce the quantities of SCFAs missing inside the gut. We designed the constructs for Acetate, Propionate & Butyrate producing enzymes.
The Acetate production enzymes pta and ack catalyze the conversion from Acetyl CoA to Acetate. Concurrently with Acetate production ATP is produced.
ATP activates the negative feedback loop of which acs is responsible (Enjalbert, B.et al. 2017). For high yield production we chose a Δacs strain.

Figure 1: The arabinose inducible Acetate production module, utilizing AckA and Pta.

Figure 2: The metabolic pathway of Acetate production.

The propionate-producing genetic construct (BBa_K3866031) consists of the three enzymes: Sbm (BBa_K3866023), YgfG (BBa_K3866024) & YgfH (BBa_K3866025). These enzymes catalyze the conversion of succinyl-CoA into propionate (Akawi et al, 2015). For high yield production of propionate and butyrate we chose an ΔldhA strain. The lack of ldhA, increases the carbon flux by promoting the anaerobic metabolism instead of the aerobic one.

Figure 3: The arabinose inducible Propionate production module, utilizing sbm, ygfG and ygfH.

Figure 4: The metabolic pathway of Propionate production.

Butyrate is produced by nine enzymes that catalyze the formation of butyrate from acetyl coA. (Miscevic, D. et al. 2019). The genes coding for those enzymes were cloned into two operons: BktB, Hbd, PhaJ, Ptb, Buk and PhaA, PhaB, Crt and Ter (BBa_K3866015).

Figure 5: The arabinose inducible Butyrate production module, utilizing the two operons.

Figure 6: The metabolic pathway of Butyrate production.

There were two main iterations on the SCFA production modules. The first one was to change the orientation of our constructs from reverse to forward as we also did for the Tango module. The second one was to replace our initial promoters with arabinose inducible ones, araBAD, converting our expression system from a constitutive to an inducible one.

References

1. Akawi L, Srirangan K, Liu X, Moo-Young M, Perry Chou C. (2015) Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol. Jul;42(7):1057-72.https://doi.org/10.1007/s10295-015-1627-4


2. Enjalbert, B., Millard, P., Dinclaux, M., Portais, J., & Létisse, F. (2017). Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway. Scientific Reports, 7(1). doi: 10.1038/srep42135.


3. Miscevic, D., Srirangan, K., Kefale, T., Abedi, D., Moo-Young, M., & Chou, C. P. (2019). Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli. Applied microbiology and biotechnology, 103(13), 5215–5230.https://doi.org/10.1007/s00253-019-09815-x


4. Pontrelli, S., Chiu, T. Y., Lan, E. I., Chen, F. Y., Chang, P., & Liao, J. C. (2018). Escherichia coli as a host for metabolic engineering. Metabolic engineering, 50, 16–46.https://doi.org/10.1016/j.ymben.2018.04.008

Cumate kill-switch module

Figure 1: The mechanism of cumate gene switch (Seo SO, Schmidt-Dannert C.2019).

Safety is of major importance for us, considering that we deal with genetically modified bacteria. That’s why one of our kill-switches focuses on killing the bacteria in a controlled manner even before exiting the gut. The cumate inducible kill-switch consists of a repressor, CymR, a cumate operator, CuO, and a toxin-antitoxin system, mazE-mazF (Choi Y. et al. 2010). In the absence of Cumate, CymR binds to the cumate operator and suppresses the toxin mazF. In the presence of Cumate, CymR loses its affinity to CuO, the toxin is expressed and the bacteria die. At first, we designed our constructs with level 0 interchangeable parts with the GoldenBraid assembly. However, if we assembled a transcription unit with the level 0 toxin part, our bacteria would die. So, we redesigned our module in order to assemble all transcription units simultaneously, containing both the antitoxin and the repressor.

References

1. Choi, Y. J., Morel, L., Le François, T., Bourque, D., Bourget, L., Groleau, D., Massie, B., & Míguez, C. B. (2010). Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Applied and environmental microbiology, 76(15), 5058–5066. https://doi.org/10.1128/AEM.00413-10


2. Seo, S. O., & Schmidt-Dannert, C. (2019). Development of a synthetic cumate-inducible gene expression system for Bacillus. Applied microbiology and biotechnology, 103(1), 303–313.https://doi.org/10.1007/s00253-018-9485-4

Εxtracellular IPTG stability

We started off our modeling from building and simulating the lac operon module, the most complex and challenging part of our design. It is an IPTG (Isopropyl β-D-1-thiogalactopyranoside) induced genetic mechanism in which we make a clear distinction between intra- and extracellular IPTG. Extracellular IPTG is the component that we used to control in order to induce and test the system. After some experiments that exhibited instability, we realized that extracellular IPTG had to be a fixed value during the simulations, as it practically is in vitro; by integrating that in our model and re-running the simulations we ended up having a smoother, more precise outcome.

Figure 1: The lac operon modified, “open” genetic circuit.

References

1. Stamatakis, M., & Mantzaris, N. V. (2009). Comparison of deterministic and stochastic models of the lac operon genetic network. Biophysical Journal, 96(3), 887–906. https://doi.org/10.1016/j.bpj.2008.10.028 https://doi.org/10.1016/j.bpj.2008.10.028

Tyrosine as lac operon’s output

At the first “open circuit” version of lac operon, its output species was GFP, which served as a general-purpose reporter gene. After looking at some literature, we noticed that GFP’s kinetics were very hard to depict, since the extraction of a kinetic formula for GFP expression always results from experimental data fitting and is often characterized by noisy dynamics. This is why we replaced GFP with Tyrosine. By doing so, we express an output that is chemically compatible with the Reporter module by using a species that is much more likely to follow the operon’s kinetics. Note that having GFP as an output did not get in the way of having an effective simulation, but was forcing the assumption that GFP follows the lac operon’s kinetics, which was rather unrealistic.

Figure 1: The Reporter Module genetic circuit.

References

1. Vanarsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G. F., Van Maris, A. J. A., & Bentley, W. E. (2020). A coculture based tyrosine-tyrosinase electrochemical gene circuit for connecting cellular communication with electronic networks. ACS Synthetic Biology, 9(5), 1117–1128.https://doi.org/10.1021/acssynbio.9b00469

Hardware

Overview
The Design Build Test Learn cycle is embedded in scientific research and naturally was in ours too. We began this year with an already designed capsule. The goal of this year was to improve upon that design and implement new ideas that we had. We improved the design by reducing the size of the capsule and thus making it safer and easier to swallow. Next we implemented our new idea of data transmittance, which used an SoC. Finally we made one last safety addition to the capsule, by implementing a mechanical kill switch.

Iteration 1. Processor and Antenna
The hardware side of the project is mainly divided into 3 major DBTL (design build test learn) cycles. The first iteration had to do with the change in the “brains” of our system. Last year we implemented the processing of our data with a simple PIC18F microprocessor. This year we decided to change that as we had greater needs in terms of data processing. After diligent research we ended up on the DA14531 BLE SoC.
This SoC gave us the ability to combine into a single tiny device both the processing and the transfer of the data as it is equipped with a bluetooth antenna that uses AES/CCM (Advanced Encryption Standard) encryption to safely transmit the data. In comparison with last year when we had temporarily implemented a simple RF transmitter, this year we wanted to connect our capsule to a phone so we had to find a different solution. We concluded on the DA14531 BLE SoC, which also allowed us to shrink down the capsule.

Iteration 2. Capsule dimensions
Taking advantage of the newly available space, our first sub iteration was to add another battery to the already existing ones, with the goal, of course, to increase the battery life of the system. The ADC consumes approximately 50 to 60 μA. Our first battery array provided 45mA so our battery life was approximately 750 hours under ideal conditions. With the third battery we boosted that to 1500 hours. After those iterations we still had space available, so we decided to shrink the capsule dimensions and specifically on the X axis. The capsule ended up being 15mm in length from the original 22m and with the addition of 1 battery.

Figure 1: The 3D design of the capsule showcasing the outer cell and the components

Iteration 3. Kill switch
Finally, we designed a mechanical kill switch that would facilitate the cell death induced by the biological arabinose inducible kill-switch. This device is located above the bacteria vat and it gets activated when a certain amount of pressure is applied on the capsule. The amount of pressure required for the activation, is equal to the breaking point that the outer cell of the capsule. The L-arabinose, that is contained in this separate space, gets mixed with the bacteria, enabling the expression of the toxin ,and the consequent death of the cells.

References

1. Zhao, K., Yan, G., Lu, L., & Xu, F. (2015). Low-Power Wireless Electronic Capsule for Long-Term Gastrointestinal Monitoring. Journal of Medical Systems, 39(2). https://doi.org/10.1007/s10916-015-0211-9


2. Zhuang, Y., Hou, W., Zheng, X., Wang, Z., Zheng, J., Pi, X., Cui, J., Jiang, Y., Qian, S., & Peng, C. (2011). A MEMS-based electronic capsule for time-controlled drug delivery in the alimentary canal. Sensors and Actuators, A: Physical, 169(1), 211–216. https://doi.org/10.1016/j.sna.2011.05.024

igem.thessaly@gmail.com