iGEM 2020 — Team GreatBay_SZ

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GreatBay SZ/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GreatBay SZ/Engineering

[![](https://static.igem.org/mediawiki/2020/b/b6/T--GreatBay_SZ--NAVBARLOGO.png)](https://2020.igem.org/Team%3AGreatBay_SZ)

* Project

  + [Description](https://2020.igem.org/Team%3AGreatBay_SZ/Description)
  + [Engineering Success](https://2020.igem.org/Team%3AGreatBay_SZ/Engineering)
  + [Proof Of Concept](https://2020.igem.org/Team%3AGreatBay_SZ/Proof_Of_Concept)
  + [Modelling](https://2020.igem.org/Team%3AGreatBay_SZ/Model)
  + [Proposed Implementation](https://2020.igem.org/Team%3AGreatBay_SZ/Implementation)
  + [Contribution](https://2020.igem.org/Team%3AGreatBay_SZ/Contribution)
* Entrepreneurship

  + [Entrepreneurship](https://2020.igem.org/Team%3AGreatBay_SZ/Entrepreneurship)
* Parts

  + [Parts](https://2020.igem.org/Team%3AGreatBay_SZ/Parts)
  + [Part Collection](https://2020.igem.org/Team%3AGreatBay_SZ/Part_Collection)
* Human Practice

  + [Integrated](https://2020.igem.org/Team%3AGreatBay_SZ/Human_Practices)
  + [Public Education](https://2020.igem.org/Team%3AGreatBay_SZ/Education)
  + [Public Engagement](https://2020.igem.org/Team%3AGreatBay_SZ/Public_Engagement)
* Lab

  + [Lab Notes](https://2020.igem.org/Team%3AGreatBay_SZ/Lab_Notes)
  + [Protocols](https://2020.igem.org/Team%3AGreatBay_SZ/Protocols)
  + [Safety](https://2020.igem.org/Team%3AGreatBay_SZ/Safety)
* Team

  + [Members](https://2020.igem.org/Team%3AGreatBay_SZ/Members)
  + [Attributions](https://2020.igem.org/Team%3AGreatBay_SZ/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AGreatBay_SZ/Collaborations)
* Poster

  + [Poster](https://2020.igem.org/Team%3AGreatBay_SZ/Poster)
* Awards

  + [Awards](https://2020.igem.org/Team%3AGreatBay_SZ/Awards)

Stack Multipurpose HTML Template

![](https://static.igem.org/mediawiki/2020/1/17/T--GreatBay_SZ--EngieeringSUCCESS.png)

#### Index

* [Overview](#overview)
* [Expression Of Protein Nanowire BIOT-WT](#Expression)
* [Generating Electricity](#Generating Electricity)
* [References](#reference)

### 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.](https://2020.igem.org/Team%3AGreatBay_SZ/Proof_Of_Concept)

### **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).

![](https://static.igem.org/mediawiki/2020/6/62/T--GreatBay_SZ--Figure1_The_Genetic_circuit_of_BIOT.png)

###### Figure 1: The Genetic circuit of BIOT

###### Table 1: The composition of BIOT Genetic Circuits

![](https://static.igem.org/mediawiki/2020/0/0f/T--GreatBay_SZ--Table_1-_The_composition_of_BIOT_Genetic_Circuits.png)

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).

![](https://static.igem.org/mediawiki/2020/d/df/T--GreatBay_SZ--Figure_3-_CRISPR-Cas3_system_for_the_deletion_of_fimA.png)

###### 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).

![](https://static.igem.org/mediawiki/2020/d/df/T--GreatBay_SZ--Figure_4-_The_PCR_verification_of_the_deletion_of_fimA.png)

###### 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).

![](https://static.igem.org/mediawiki/2020/c/c8/T--GreatBay_SZ--Figure_5-_The_procedures_for_BIOT_nanowires_purification.png)

###### Figure 4: The procedures for BIOT nanowires purification.

![](https://static.igem.org/mediawiki/2020/4/44/T--GreatBay_SZ--Figure_6-_The_Protein_Concentration_of_BIOT_purification.png)

###### 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).

![](https://static.igem.org/mediawiki/2020/5/5f/T--GreatBay_SZ--TFigure_2-_The_Structure_of_e-PN_monomer_protein.png)

###### 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]

![](https://static.igem.org/mediawiki/2020/a/a9/T--GreatBay_SZ--Figure_7-_BIOT_membrane_on_the_transparent_membrane.png)

###### 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](https://2020.igem.org/Team%3AGreatBay_SZ/Human_Practices)). 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.

![](https://static.igem.org/mediawiki/2020/1/15/T--GreatBay_SZ--Figure_8-_BIOT_Assembly_and_Measurement.png)

###### 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).

![](https://static.igem.org/mediawiki/2020/f/fe/T--GreatBay_SZ--data1.png)

###### 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.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GreatBay SZ/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GreatBay SZ/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)