Team:GreatBay SZ/Poster
Poster: GreatBay_SZ
BIOT: Biotechnology-Driven Internet of Things
Produced By GreatBay_SZ 2020
Inspiration
IoT, or the Internet of Things, refers to the network of physical objects capable of gathering and sharing electronic information. In the foreseeable 2030, there will be approximately 125 billion IoT devices; every second, 127 devices are being connected to the network.
However, to power devices within the expanding IoT network is a critical issue. Current energy sources, including lithium and primary batteries or environmental energy, have some fundamental drawbacks regarding their sustainability, lifespan, or rigid environmental requirements. As a result, we need a self-sustained, environmentally-friendly, and highly adaptive solution capable of powering IoT devices.
Our inspiration came from the high humidity in Shenzhen. Throughout the whole year, the humidity of Shenzhen remains above 70%; What's more, over 70% of the globe has humidity that's higher than 50%. The omnipresent atmospheric moisture could provides an alternative for continuous and unlimited energy production.
However, to power devices within the expanding IoT network is a critical issue. Current energy sources, including lithium and primary batteries or environmental energy, have some fundamental drawbacks regarding their sustainability, lifespan, or rigid environmental requirements. As a result, we need a self-sustained, environmentally-friendly, and highly adaptive solution capable of powering IoT devices.
Our inspiration came from the high humidity in Shenzhen. Throughout the whole year, the humidity of Shenzhen remains above 70%; What's more, over 70% of the globe has humidity that's higher than 50%. The omnipresent atmospheric moisture could provides an alternative for continuous and unlimited energy production.
Introduction
A self-sustained, environmentally-friendly, and highly adaptive solution capable of powering billions of IoT devices in urgently needed. Inspired by widespread humidity in our planet, we developed a moisture-driven energy harvesting technology: BIOT, or Biotechnology Driven Internet of Things.
The core of BIOT is optimized electrically conductive protein nanowires (e-PNs), containing nanopores and surface carboxyl groups that enable power generation through water molecules in the air[1].
In this project, we successfully fabricated our BIOT, achieved high-efficient protein production, improved BIOT power efficiency, and designed hardware for customized settings. The following is a summary of the core features of BIOT：
As far as we know, this will be the first time that biotechnology has been applied in the field of IoT. With BIOT, we can see a grand future where hundreds of billions of IoT devices connected the whole world.
The core of BIOT is optimized electrically conductive protein nanowires (e-PNs), containing nanopores and surface carboxyl groups that enable power generation through water molecules in the air[1].
Fig.1 G.sulfurreducens and the electrically conductive protein nanowires it produces[1]
In this project, we successfully fabricated our BIOT, achieved high-efficient protein production, improved BIOT power efficiency, and designed hardware for customized settings. The following is a summary of the core features of BIOT：
Fig.2 The summary of the core features of BIOT
As far as we know, this will be the first time that biotechnology has been applied in the field of IoT. With BIOT, we can see a grand future where hundreds of billions of IoT devices connected the whole world.
Successful Production of BIOT
Electrically-conductive Protein Nanowires (e-PNs) is the core of our project. However, it’s origin in G. sulfurreducens post limitations in producing e-PNs due to the high growing-condition requirements. Alternatively, Escherichia coli can be an ideal chassis for e-PN fabrication, as a common platform for the commercial-scale production of organic commodities with the rich toolboxes for tuning gene expression. Thus, we followed the design of genetic circuits on an ACS Synthetic Biology paper[2], which was composed of 2 modules: The protein nanowire monomer and the Type IV secretion system.
We verified that BIOT's protein monomers were successfully expressed in E. coli using His antibody against the C-terminus His tag of monomer protein in western blot.
Before transferring the Type IV secretion system to E. coli Top10, we had to remove the Type I system. If the two systems were all present, the assembly efficiency of e-PNs would be affected. FimA was the key gene in the original Type I system. We successfully removed the original Type I system within the E.coli by using the CRISPR-Cas9 system to knocked off the fimA gene — the key gene of Type I system.
We then successfully introduced the constructed expression plasmid into the fimA-knockout E. coli. We followed the protocol[2] to obtain purified protein from the E.coli, with a concentration of about 200 to 400 ng/μl.
Fig.1 The Genetic circuit of BIOT
We verified that BIOT's protein monomers were successfully expressed in E. coli using His antibody against the C-terminus His tag of monomer protein in western blot.
Fig.2 The X-ray Structure of e-PN monomer protein (a) and Western blot to verify the expression of BIOT monomers (b).
Before transferring the Type IV secretion system to E. coli Top10, we had to remove the Type I system. If the two systems were all present, the assembly efficiency of e-PNs would be affected. FimA was the key gene in the original Type I system. We successfully removed the original Type I system within the E.coli by using the CRISPR-Cas9 system to knocked off the fimA gene — the key gene of Type I system.
Fig.3 CRISPR-Cas9 system[3] for the deletion of fimA gene in E. coli Top10
Fig.4 The gRNA sequence used in our project (a) and the PCR verification of the deletion of fimA in E. coli Top10 using CRISPR-Cas9 system (b).
We then successfully introduced the constructed expression plasmid into the fimA-knockout E. coli. We followed the protocol[2] to obtain purified protein from the E.coli, with a concentration of about 200 to 400 ng/μl.
Fig.1 The procedures for BIOT nanowires purification. (a) Cell culture; (b) Nanowires blending; (c) Nanowires filtration; (d) Protein concentration measurement.
Fig.2 BIOT nanowires were filtrated on a 100kDa ultrafiltration membrane (a) and the Protein Concentration of BIOT purification (b).
Valid Measurement of BIOT's Output
After successfully purified the nanowires, we fabricated the basic module of BIOT using a large positive gold electrode at the bottom and a tiny circular carbon negative electrode on the top.
Under the relative humidity (RH) of 60%, we efficaciously gained output voltage of 0.35V and current fluctuating from 0.2μA to 2μA with the BIOT-WildType protein. Additionally, to find the optimum relative humidity for the battery to function, we did the measurement with different relative humidities. The optimum RH was roughly at 40-50%, where the voltage rises to about 0.42V.
Fig.1 BIOT Assembly and Measurement. (a) The basic parameter of the basic BIOT module; (b) Different ways for BIOT electric generator assembly; (c) Digital multimeter, Keithley 2401 was used for Voltage and Current measurement.
Under the relative humidity (RH) of 60%, we efficaciously gained output voltage of 0.35V and current fluctuating from 0.2μA to 2μA with the BIOT-WildType protein. Additionally, to find the optimum relative humidity for the battery to function, we did the measurement with different relative humidities. The optimum RH was roughly at 40-50%, where the voltage rises to about 0.42V.
Fig.2 The Key parameters of BIOT basic module. (a) Voltage; (b) Current; (c) Voltage under different humidity conditions;
Reliable Hardware Design
1. Connect more, achieve higher.
To further increase the output voltage, we made a board that can be used to connect multiple batteries in series, with the designed copper sheet structure on top of it. We successfully gained an output of 1.6V with four modules, which confirmed the feasibility of increasing the output voltage in this way.
2. Stable the output for more scenarios.
Power management integrated circuits (PMIC) are integrated circuits for power management. The environmental energy itself has the characteristics of unstable power output, and the PMIC module specially developed for environmental energy technology (such as BQ25570 or LTC3588) can store and manage the energy from the environment, and output electrical energy in a more stable way.
In our design of the circuit, PMIC temporarily stores BIOT's output power through its power management chip and thus, powers the IOT devices stably.
We then tested the PMIC module to verify that it could work as we expected, managing the electromotive force. The PMIC module will further enable BIOT to meet the needs of more scenarios.
To further increase the output voltage, we made a board that can be used to connect multiple batteries in series, with the designed copper sheet structure on top of it. We successfully gained an output of 1.6V with four modules, which confirmed the feasibility of increasing the output voltage in this way.
Fig.1: (a & b) The board with the designed copper sheet structure on top of it; (c) Connected nanowire devices in series lead to a higher voltage output.
Fig.2 The photo of voltmeter which was connected in parallel with 4 BIOT modules
2. Stable the output for more scenarios.
Power management integrated circuits (PMIC) are integrated circuits for power management. The environmental energy itself has the characteristics of unstable power output, and the PMIC module specially developed for environmental energy technology (such as BQ25570 or LTC3588) can store and manage the energy from the environment, and output electrical energy in a more stable way.
In our design of the circuit, PMIC temporarily stores BIOT's output power through its power management chip and thus, powers the IOT devices stably.
Fig.3 Using PMIC module to stable the BIOT output
We then tested the PMIC module to verify that it could work as we expected, managing the electromotive force. The PMIC module will further enable BIOT to meet the needs of more scenarios.
Fig.4 The circuit containing a PMIC module and a capacitor lighting up a LED
Optimization of BIOT's Power Efficiency
We engineered the protein monomer of e-PNs to increase the voltage level. The two methods used are below.
1. Increasing the number of carboxyl groups
We established mathematics models based on the one in the original research on Nature[1] and found out that when the relative humidity is at the same level, the potential difference is directly proportional to the number of carboxyl groups.
To verify our idea, we designed 16 mutants of the e-PN gene, to increase the number of carboxyl groups which reacted with water molecules to form hydrogen ions. We made it by replacing 1 to 4 amino acids in non-conservative regions with Aspartic acid or Glutamic acid since they have two carboxyl groups.
The amino acids we chose to replace had similar structures to them based on Grantham's distance Model and some other methodologies[3-12].
2. Decreasing the diameter
By decreasing the diameter, it becomes harder for the water molecules to pass through the nanopores; it will lead to a greater water potential gradient as reported from the work published on Nature. Through literature search, we found two mutations F51W and Y57W can make the diameter of nanowires decrease from 3nm to 1.5nm[13].
3. Result
Before practical measurement, we first built homologous models to predict the structure of mutated proteins; we then analyzed the Root-mean-square deviation of atomic positions value (RMSD) between BIOT-WT and mutants using PyMOL. The smaller the value is, the more similar they are. The results were all below 0.03, which was far lower than the threshold.
By decreasing the diameter of the nanowires to decrease the diameter of the nanopores, we gained an output voltage up to 0.47V with the room relative humidity of about 60%. By increasing the number of carboxyl groups in protein monomer, we enhanced the voltage up to around 0.51V with BIOT-4A, by 46% compared with the 0.35V of BIOT-WT.
1. Increasing the number of carboxyl groups
We established mathematics models based on the one in the original research on Nature[1] and found out that when the relative humidity is at the same level, the potential difference is directly proportional to the number of carboxyl groups.
Fig.1 The model for BIOT potential generation
To verify our idea, we designed 16 mutants of the e-PN gene, to increase the number of carboxyl groups which reacted with water molecules to form hydrogen ions. We made it by replacing 1 to 4 amino acids in non-conservative regions with Aspartic acid or Glutamic acid since they have two carboxyl groups.
Fig.2 Aspartic acid (Asp) and Glutamic acid (Glu) both have extra -COOH.
The amino acids we chose to replace had similar structures to them based on Grantham's distance Model and some other methodologies[3-12].
Fig.3 The mutant sites of mutants with 1-4 Carboxylic groups added
2. Decreasing the diameter
By decreasing the diameter, it becomes harder for the water molecules to pass through the nanopores; it will lead to a greater water potential gradient as reported from the work published on Nature. Through literature search, we found two mutations F51W and Y57W can make the diameter of nanowires decrease from 3nm to 1.5nm[13].
Fig.4 Increase the potential value of BIOT by decreasing the diameter of nanowires
3. Result
Before practical measurement, we first built homologous models to predict the structure of mutated proteins; we then analyzed the Root-mean-square deviation of atomic positions value (RMSD) between BIOT-WT and mutants using PyMOL. The smaller the value is, the more similar they are. The results were all below 0.03, which was far lower than the threshold.
Fig.5 The RMSD values of the 17 mutants comparing with the BIOT-WT protein
Fig.6 The homologous models of BIOT-WT and the 17 mutants
By decreasing the diameter of the nanowires to decrease the diameter of the nanopores, we gained an output voltage up to 0.47V with the room relative humidity of about 60%. By increasing the number of carboxyl groups in protein monomer, we enhanced the voltage up to around 0.51V with BIOT-4A, by 46% compared with the 0.35V of BIOT-WT.
Fig.7 The output voltage of the 17 mutants comparing with the BIOT-WT; the graph at the upper right corner was the data gained in the repetitive experiment, where the gap between the output of mutants and wildtype narrowed.
Optimization of BIOT's Productivity
To reach further commercialization, we then targeted the RBSs of the Type IV e-PNs assembly system. We aimed at increasing the number of type IV secretion machines, thus optimizing the productivity of the e-PNs, then lowering the cost. We chose the RBS of hofB, hofM and ppdA, whose sequence changes were reported to affect the expression of nanowires[2].
We originally planned to create a library for each RBS, then randomly combine them to find the optimum combination. But we were forced to reduce the size of our RBS library down to 3*2*3 since we couldn't find an effective method to screen the best combination out of the 1000 possibilities we envisioned.
Due to time constrain, we only managed to successfully introduce six combinations, but we still obtained some optimistic results. We successfully enhanced the productivity of BIOT, with the optimum combination RBS4 we designed. The productivity was increased by 34.1%, from 346ng/μl to 465ng/μl.
We originally planned to create a library for each RBS, then randomly combine them to find the optimum combination. But we were forced to reduce the size of our RBS library down to 3*2*3 since we couldn't find an effective method to screen the best combination out of the 1000 possibilities we envisioned.
Fig.1 (a) Three RBS was selected for library construction; (b) The sequence and Translation initial strength (T.I.R) of wildtype RBS for three genes; (c) The scheme of the library size and strength of three RBS
Fig.2 The 3*2*3 RBS library combinations (a) and the sequence of each designed RBS (b)
Due to time constrain, we only managed to successfully introduce six combinations, but we still obtained some optimistic results. We successfully enhanced the productivity of BIOT, with the optimum combination RBS4 we designed. The productivity was increased by 34.1%, from 346ng/μl to 465ng/μl.
Fig.3 RBS optimization increases the production of BIOT (a) and the RBS combinations for each optimized mutant
Human Practices
Integrated HP and Entrepreneurship
Market Targeting：What’s in need?IoT sensors in traditionally inaccessible locations like desolate villages or challenging mountains is one of the significant fortes of IoT devices that requires sustainable and green batteries.
Business Plan
· Our entrepreneurial development is based on several revisions on our business plan, given advise and intructions from technical and marketing experts.
· Research + Planning → First Draft → Technical Advice → First Revision→ Feedback on Marketing and Product Prototype from IoT Companies → Second and Third Revisions → Evaluations on Business Module from Investment Experts → Last Revision → Final Business Plan
Fig.1 Revenue and cost structure in our business plan
Exhibition
· Aiming at gaining feedback from business experts, we attended the International Internet of Things Exhibition 2020 (IOTE) in Shenzhen and received suggestions on future application and investment directions.
Fig.2 Team members Shiyun Liang, Weiyu Zhang, Yutong Cai, Wanmi Sui and Feiyang Hu on the IOTE
Science Communication
1. Creating a fascinating online game about CRISPR in the form of an interactive simulation
2. Producing an educational video onto Bilibili(Chinese online video sharing platform), to introduce synthetic biology and various gene-editing technologies
3. Holding a teaching activity about CRISPR which consisted of a lecture and an interactive paper-cutting game
Fig.4 Team members Ruijia Sun and Jiaxin Li instructing the listeners to create the paper model of CRISPR
Collaboration
HFI: Providing instruction and procedure of knocking out a gene using CRISPR
SZU-China: Contributing to their pamphlet about contemporary iGEM projects and fundamental knowledge of Synthetic Biology
GreatBay_SCIE: Gaining advice on wiki construction
Fig.5 Our team members Weiyu Zhang, Yutong Cai and HFI's team members
Summary
What we have achieved:
1. Successful expression of e-PNs
2. Valid measurement of output: 0.35V and 0.2-2mA
3. Optimization of BIOT's productivity: 465ng/μl by 34.1%
4. Optimization of BIOT's output voltage: 0.51V by 46%
5. Test of hardware design for real-life applications
6. Exploration for commercialization of BIOT in the market of IoT
7. Promotion of Synthetic Biology and iGEM to the neighbourhood
What we plan to do in the future:
1. Repeat experiments with various data sets for a more accurate result
2. Continue exploration on optimization of the protein
3. Further develop circuit design & Power real-life wireless devices
4. Subsequent industrialization: refinement on business plan & large-scale production
Acknowledgement
Support from Others:
Wang Weixu - For guiding us in mathematical modelling in Matlab.
Zong Yeqing - For giving us advice on measurement and building hardware.
Chen Yiming - For filming all our recordings and capturing moments.
Lv Wei (MROBOT) - For giving us advice on measurements of voltage and currents.
Fanxin Meng (Xidian University) - For giving us advice on measurements of voltage and currents.
Xu Jiaqi, Zhao He - For providing us with space and helping us schedule our education events.
Zhao Xuanyu - For assisting us answering questions from the audience during the CRISPR event.
Zhang Yihao - For showing up in our promotion video.
Feng Zhiye - For carrying out our logo with our ideas.
Shan Jiang - For helping us improve wiki figures and content.
Zhou Zhengkang, Li Jiayang - For supporting us in biological lab work.
Zhang Haoqian, Li Shiyuan, Li Cheng - For giving us suggestions for our business plan.
Schools and Sponsors:
Reference
1. Liu, Xiaomeng, et al. “Power Generation from Ambient Humidity Using Protein Nanowires.” Nature, vol. 578, no. 7796, Feb. 2020, pp. 550–554, 10.1038/s41586-020-2010-9.
2. Ueki, Toshiyuki, et al. “An Escherichia Coli Chassis for Production of Electrically Conductive Protein Nanowires.” ACS Synthetic Biology, vol. 9, no. 3, 3 Mar. 2020, pp. 647–654, 10.1021/acssynbio.9b00506.
3. Dagan, Tal, et al. “Ratios of Radical to Conservative Amino Acid Replacement Are Affected by Mutational and Compositional Factors and May Not Be Indicative of Positive Darwinian Selection.” Molecular Biology and Evolution, vol. 19, no. 7, 1 July 2002, pp. 1022–1025, academic.oup.com/mbe/article/19/7/1022/1068565, 10.1093/oxfordjournals.molbev.a004161. Accessed 27 Sept. 2020.
4. EPSTEIN, CHARLES J. “Non-Randomness of Ammo-Acid Changes in the Evolution of Homologous Proteins.” Nature, vol. 215, no. 5099, July 1967, pp. 355–359, 10.1038/215355a0. Accessed 27 Sept. 2020.
5. Grantham, R. “Amino Acid Difference Formula to Help Explain Protein Evolution.” Science (New York, N.Y.), vol. 185, no. 4154, 1974, pp. 862–4, www.ncbi.nlm.nih.gov/pubmed/4843792, 10.1126/science.185.4154.862. Accessed 22 Jan. 2020.
6. Graur, Dan. “Amino Acid Composition and the Evolutionary Rates of Protein-Coding Genes.” Journal of Molecular Evolution, vol. 22, no. 1, Aug. 1985, pp. 53–62, 10.1007/bf02105805. Accessed 27 Sept. 2020.
7. ---. Molecular and Genome Evolution. Sunderland, Massachusetts, Sinauer Associates, Inc, 2016.
8. Miyata, Takashi, et al. “Two Types of Amino Acid Substitutions in Protein Evolution.” Journal of Molecular Evolution, vol. 12, no. 3, Mar. 1979, pp. 219–236, 10.1007/bf01732340. Accessed 27 Sept. 2020.
9. Sneath, P.H.A. “Relations between Chemical Structure and Biological Activity in Peptides.” Journal of Theoretical Biology, vol. 12, no. 2, Nov. 1966, pp. 157–195, 10.1016/0022-5193(66)90112-3. Accessed 27 Sept. 2020.
10. Xuhua Xia. Data Analysis in Molecular Biology and Evolution. Boston, Kluwer Academic Publishers, 2001.
11. Yampolsky, Lev Y., and Arlin Stoltzfus. “The Exchangeability of Amino Acids in Proteins.” Genetics, vol. 170, no. 4, 1 Aug. 2005, pp. 1459–1472, www.ncbi.nlm.nih.gov/pmc/articles/PMC1449787/, 10.1534/genetics.104.039107. Accessed 10 June 2020.
12. Zuckerkandl, Emile, and Linus Pauling. “Molecules as Documents of Evolutionary History.” Journal of Theoretical Biology, vol. 8, no. 2, Mar. 1965, pp. 357–366, 10.1016/0022-5193(65)90083-4. Accessed 29 Feb. 2020.
13. Tan, Yang, et al. “Synthetic Biological Protein Nanowires with High Conductivity.” Small, vol. 12, no. 33, 13 July 2016, pp. 4481–4485, 10.1002/smll.201601112.