Team:GreatBay SZ/Engineering
Overview
The aim of GreatBay_SZ 2020 is to develop BIOT, an electric generator harvesting energy from moisture in the air to solve the power-supply issues billions of IoT devices are facing. In this Engineering Success section, we are going to demonstrate the success of the project from the perspective of genetic circuits design and BIOT electricity generation. In short, we successfully designed and constructed genetic circuits for BIOT nanowires expression and made BIOT produce a stable and significant voltage and current, which is the basis for work in this summer. See more about our project, please read Proof-of-Concept page.
Expression Of Protein Nanowire BIOT-WT
We successfully constructed the BIOT-WT plasmid according to the genetic pathway provided by a paper we found [1]. This gene circuit composed of two parts. The first part is the protein monomer. We fused a signal peptide to the N-terminus of the protein monomer, which can help the protein to be secreted outside the cell membrane.The second part is the Type IV secretion system. It is a secreted protein machine distributed on the cell membrane, which is used to assemble BIOT's protein monomers into nanowires that can be up to 20 microns in length.(figure1;table1).
Figure 1: The Genetic circuit of BIOT
Table 1: The composition of BIOT Genetic Circuits
Before transferring 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 of e-PNs would be affected. FimA was the key gene in the original Type I system, so we got rid of it with CRISPR and a donor DNA as template (figure 2).
Figure 2: CRISPR-Cas3 system for the deletion of fimA gene in E. coli Top10.
The success deletion of filmA form E. coli Top10 was verified by PCR (figure 3).
Figure 3: The PCR verification of the deletion of fimA in E. coli Top10 using CRISPR.
Then we followed the protocol to purify the target protein(figure 4), gaining protein with a concentration ranging from 200 to 400 μg/ml(figure 5).
Figure 4: The procedures for BIOT nanowires purification.
Figure 5: The Protein Concentration of BIOT purification.
Western Blot was used to confirm the product protein as our targeting one, and the result showed it clearly (figure 6).
Figure 6: The Structure of e-PN monomer protein(A) and Western blot to verify the expression of BIOT monomers;(B).
Generating Electricity
We made a protein film following the instructions from the paper we researched(figure 7).[2]
Figure 7: BIOT membrane on the transparent membrane.
After that, we struggled a bit to produce electricity. We looked for help from some experts in the realm of electrical engineering in China and the author of the original paper(See our Human Practices for details). Through this continuous process of experimenting and debugging, we concluded that the number of protein layers, the proper pH(about 2), and the material of the electrode(Pt，Au or C) were crucial for the generation of electricity. Then we successfully built up the basic module of BIOT (figure 8).The gold positive electrode at the bottom is larger so that the e-PNs can be positioned on it. And the tiny circular carbon piece works as a negative electrode on the top.
Figure 8: BIOT Assembly and Measurement
We eventually succeeded in producing stable voltage at about 0.35V with a current of 500nA or so (under 60% relative humidity)(figure 9 A,B).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 (figure 9C).
Figure 9: The Key parameters of BIOT basic module. A, The voltage of BIOT; B, the current of BIOT; C, Voltage of BIOT under different relative humidities.
References
[1] 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.
[2] Liu, Xiaomeng, et al.. Power Generation from Ambient Humidity Using Protein Nanowires. Nature, vol.578, no.7796, Feb.2020, pp.550–554.