Team:LINKS China/Poster

PICACHU: PIlin Constructed nAnowires production CHassis Unlocking ---- LINKS_China 2020
Presented by Team LINKS_China 2020

Brooklyn, Shangyi, Eve, Stephen, Johnny, Carl, Marie, Mesue, Kevin, Aaron, Leo, May, Fion, Wency, David L, David Z, Mike, Zane, Josh, Bruce, Boxiang Wang, Han Yu, Qing Liang, James, and Yoyo.

iGEM Student Team Member, iGEM Team Mentor

Abstract

Electronic conductive pili (e-pili) is an electricity conduction material produced by microbes, which has been proved for its electricity production in humid environments. Recently, various e-pili were discovered but not suitable for large-scale production due to the severe cultivation conditions and bio-safety concern. This year, LINKS_China designed PICACHU, an E.coli chassis to express different e-pili. We manufactured a pili-generator consists of 12 genes for pili assembly, expressed three pili, and created a new measurement to verify the pili production. Furthermore, we renovated the generator and optimized the growth condition of the cells to increase the yield. Ultimately, we transformed the e-pili product into a nanowire battery, and expected it to provide sustainable clean energy. We are aiming to apply PICACHU to track migrant birds, to monitor the wetland, and to trace the trash flowing movement in the ocean. Hopefully, more possibilities of e-pili application will be explored in the future.
PICACHU!
Presented by Team LINKS_China 2020

Achievements

1. Made an E-pili generating system for different E-pili
2. Produced E-pili for electricity generation
3. Achieved high productivity for E-pili
4. Designed a measuring method for detecting E-pili growth
5. Built different kinds of sustainable batteries suitable for various market needs
PICACHU!
Presented by Team LINKS_China 2020
E-pili Battery

What is E-pili Battery?
E-pili is a kind of Type IV Pili(T4P) that is capable of conducting electricity and having long-range electron exchange between itself and the external environment. Bacteria and archaea secrete these pili to decorate their surface and enable adhesion to various substrates. Here, we use e-pili to form a film around 3 μm thick, with humidity differences between its top and bottom surfaces to produce a electric potential difference, creating a bio-battery.


How does it function?
The carboxy groups on the top surface can interact with water molecules and ionize them, creating mobile protons and stationary carboxylates. The further the carboxy groups are from the surface, the less likely that they would be ionized. Then, because the proton concentration of the top is higher, the protons would diffuse downwards, creating a positive electric potential at the bottom and leaving a negative electric potential on the top. As a result, an electric potential difference is formed down the humidity gradient and a current is thus created if the top and bottom of the film are connected together.
Figure 2: schematic diagram of E-pili battery
Type IV pilus generator
The pili generating system includes 13 components: pilA and pili generator gene cluster that constitutes the rest 12 components: hofBC, hofMNOPQ, ppdAB-ygdB-ppdC, and gspO(fig. 1a, b). We designed and generated three types of E-pilin, GsPilA, GmPilA, and PaPilA in E.coli.
Figure 1: How E-pili generator operates


This year, to acquire a complete pili generating system, LINKS_China first ordered oligo DNAs from GsPilA and assembled by SOE PCR. Then we amplified each gene cluster of the pili generator from DH5a separately on pSBIC3 vector to form three sub-plasmids, P2: GsPilA and hofBC; P3: hofMNOPQ and P4: ppdAB-ygdB-ppdC and gspO. We also constructed another sub-plasmid which contains pET28a vector for the backbone. Afterward, we assembled four sub-plasmids to the final major plasmid from DH5a through Golden Gate Assembly.

Because of the better production with EHEC, we conducted point mutation on individual genes in each sub-plasmid and deriving the gene sequence to EDL933 similar. These new sub-plasmids will express the same amino acid sequence as expressed by EDL933. We discovered two new pili PaPilA and GmPilA, and we inserted them separately into different sub-plasmids.
Increasing E-pili yield

Best Cultivation Conditions Analysis
We conducted an analysis on determining the best cultivation condition for a better e-pili productivity by using our designed measurement. In this analysis, we did two repeated experiments with all pili under constant environmental conditions for cultivation except the variables: cultivation time and oxygen level. To further prove the use of the E.coli EDL933 pili generator’s function of types of pili.

According to each part of the analysis results, we summarized these points for cultivation that could let us harvest the most pili production: sealed plate with limited oxygen level; cultivation time between 48h and 72h; and pili expression with EHEC pili generator. Furthermore, we developed a new strategy of decoupling pili generator gene clusters with modification to further enlarge our pili yield, and are planning on completing this optimization in the future.
E-pili Function

To test the performance of our three types of E-pili (GsPilA, GmPilA, and PaPilA), we conducted several experiments regarding the current and voltage of them. Succinctly, we connected the multimeter to our battery for direct measurement of voltage and current. For Gs pili, the current is 0.129mA and the voltage is 0.21V for each battery. (pH=10) Under the same condition, these data for Pa and Gm pili are 7.4mA, 0.48V; 0.602mA, 0.27V respectively. During the experiments, we noticed that the pH value will influence the performance of batteries: the current is proportional to the acidity, and it reaches the peak when pH=2. Thus, we repeated our experiment under pH=2 and found that Pa pili has the best performance (60.5mA), followed by Gs pili (16.5mA) and Gm pili (11.7mA). Note that the voltages almost remain constant among the three. By connecting each battery in series, we raised the total voltage to 2.2V in circuit. On the strength of these experiments, we successfully found that Pa pili is the best candidate for battery production under the mentioned condition.
E-pili Production

Pili Harvest
For our pili expression, we chose strain E.coli BL21 because it is the most commonly used engineering strain and its T4P production ability was proved. We cultured the bacteria in solid M9 mediums with glycerol as the carbon source. After 48h cultivation at 30°C, we harvested the bacteria, conducted extraction and purification, and finally gained a pili solution in 150mM ethanol-amine after filtrating with a 100kDa membrane in nitrogen gas.


Confirmation
We ordered SEM Photography for physical confirmation of e-pili produced. we can see even distribution of e-pili and porous structures between each individuals, which is like a sponge that can assure a water potential gradient would form within the pili membrane—crucial and beneficial for e-pili to generate electricity. Besides, we confirmed that the E.coli does produce our target e-pili by conducting Western blot experiment.


Our team repeated the measurement three times and obtained the tendency of production for different pili within less time. Additionally, the most valuable meaning of this measurement is that it is not only able to detect the production of pili for bacteria, but also provide the ability to test any protein that is expressed out of the cell membrane.
Measurement
This year, LINKS_China invented a new measurement which is based on the combination of His-tags with the primary antibodies and secondary antibodies directly on the pili produced on the E.coli membrane without protein extraction and Western Blot experiment. This measurement can be used to detect every outer membrane protein on the cell.

Confirmation
Comparing with the previous methods, this measurement is quicker, clearer, and more accurate because it consists of fewer procedures and shorter periods which will help to obtain fewer errors. We measured the absorbance of samples at OD450 and divided by the value at OD600 and subtracted the negative control.


Our team repeated the measurement three times and obtained the tendency of production for different pili within less time. Additionally, the most valuable meaning of this measurement is that it is not only able to detect the production of pili from bacteria, but also provide the ability to test any protein that is expressed out of the cell membrane.
Human Practices
How PICACHU can help the natural environment
Shenzhen, as a coastal city, has a long coastline and a significant amount of wetland. However, because of a lack of research on migratory birds, which are indicators of wetland, researchers cannot monitor the wetland, leading to the disappearance of wetland. Therefore, to protect the wetland, we decided to use the battery to help scientists to study migratory birds by powering GPS. To further learn about migratory birds and GPS, we went to the Dapeng Leucadendron Floridum Wetland Park. In the park, experts were excited about the project after our introduction. They also told us that providing power to sensors, which are used to protect mangrove forests, century-old trees, and traditional villages, is a problem.


Further investigations
After learning their demands, we contacted Seeed, a large sensor company. Besides contacting with various sensors, we learned their standardization spirit there as well, which inspired us to design our AA/AAA batteries, and knew a fantastic project initialed by WWF, called Marine Litter Detective—in the shape of Poke Ball, tracing the pattern of litter by studying ocean current. But it has a fatal weakness: lithium batteries' pollution.


However, PICACHU enables the project to be a hundred percent environmentally friendly because it is biodegradable, which is significant for the ball to prevent further pollution since it cannot be retrieved after the launching. We are currently developing a flexible PCB battery, suggested by Professor Qin from Shenzhen University, designed to install on the Poke Ball's inner surface. In the future, we want to install it on the inner surface of a spaceship and power equipment there.


Public Education: Education Kit
Our team’s educational kit is designed for primary and middle school students, aiming to increase young people’s understanding of synthetic biology. The kit includes three experiments: extracting DNA from household materials, citrus batteries, and making culture medium. And all the materials, such as edible agar, clay cell models and alcohol, are safe for teenagers to use. We prepared booklets and recorded demonstration videos in order to instruct students to complete the experiments. These educational kits are distributed to different schools to motivate the young’s interests of participating in the synthetic biology.


Escape Room Game
We also designed a room escape game called “PICACHU”. The player is trapped in a laboratory and needs to make a battery of e-pili that can generate electricity to open the door and get out of the laboratory. The game contains basic experiments such as PCR and Gibson Assembly. It can help players understand the experimental procedures of our project as well as part of the synthetic biology.
Hardware
Strength of battery
To test the performance of our three types of E-pili (GsPilA, GmPilA, and PaPilA), we conducted several experiments regarding the current and voltage of them. Succinctly, we connected the multimeter to our battery for direct measurement of voltage and current. For Gs pili, the current is 0.129mA and the voltage is 0.21V for each battery. (pH=10) Under the same condition, these data for Pa and Gm pili are 7.4mA, 0.48V; 0.602mA, 0.27V respectively. During the experiments, we noticed that the pH value will influence the performance of batteries: the current is proportional to the acidity, and it reaches the peak when pH=2. Thus, we repeated our experiment under pH=2 and found that Pa pili has the best performance (60.5mA), followed by Gs pili (16.5mA) and Gm pili (11.7mA). Note that the voltages almost remain constant among the three. By connecting each battery in series, we raised the total voltage to 2.2V in circuit. On the strength of these experiments, we successfully found that Pa pili is the best candidate for battery production under the mentioned condition.

Constructs of battery
The power generating complex has four parts: the nanowire film, top electrode (negative), bottom electrode (positive), the voltage regulator. We employed series circuit to increase power output.
Nanowire film: the film of e-pili that generates an electrical potential difference by utilizing the moisture gradients inside of the film. Bottom electrode: the point of high electric potential of the circuit and the platform that the film sits on, which needs to have good conductivity.
Top electrode: the point of high electric potential of the circuit that needs to have good conductivity and sufficiently contact to the top surface of the film without compromising moisture entrance.
Voltage regulator: the electronic component in the circuit that stabilizes the voltage output on the load.

Unlike common renewable energy sources, such as wind turbines and solar panels, the nanowire film is characterized by lightweight, 100% degradable, few restrictions (need no wind or sunlight), and theoretically infinite longevity. As a result, we planned to use it to replace the batteries powering small devices such as marinedebris sensors, bird trackers, and micro-GPS, where bulky power sources and frequent recharging are not feasible.
Acknowledgement

LINKS_China would first like to express our gratitude to LINKS Academy who guided and provided us with academic support all along the way. Next, we sincerely thank SnapGene and Matlab for the sponsorships of their application softwares. Thank you Seeed for equipping us with general knowledge of sensors, providing us the opportunity to realize our battery into applications. Finally, we were honored to have KEYSTONE-2020, SEHS-China, and SZU-China as mutual learning partners.