iGEM 2021 — Team SJTang

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# Team:SJTang/Notebook

Team:SJTang - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/1/1a/T--SJTang--nav_icon.svg)](https://2021.igem.org/Team%3ASJTang)

* Background
  + [Description](https://2021.igem.org/Team%3ASJTang/Description)
  + ---
  + [Market Study](https://2021.igem.org/Team%3ASJTang/Market_Study)
  + [Policies](https://2021.igem.org/Team%3ASJTang/Policies)
* Project
  + [Design](https://2021.igem.org/Team%3ASJTang/Design)
  + [Results](https://2021.igem.org/Team%3ASJTang/Results)
  + [Engineering](https://2021.igem.org/Team%3ASJTang/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ASJTang/Contribution)
  + [Model](https://2021.igem.org/Team%3ASJTang/Model)
  + [Proof of Concept](https://2021.igem.org/Team%3ASJTang/Proof_Of_Concept)
  + [SDGs](https://2021.igem.org/Team%3ASJTang/Sustainable)
  + [Safety](https://2021.igem.org/Team%3ASJTang/Safety)
  + [Graphic Design](https://2021.igem.org/Team%3ASJTang/Graphic_Design)
* Hardware
  + [Overview](https://2021.igem.org/Team%3ASJTang/Hardware)
  + ---
  + [Machinery](https://2021.igem.org/Team%3ASJTang/Machinery)
  + [Software](https://2021.igem.org/Team%3ASJTang/Software)
  + [Interview](https://2021.igem.org/Team%3ASJTang/Hardware_Interview)
* Human Practice
  + [Overview](https://2021.igem.org/Team%3ASJTang/Human_Practices)
  + ---
  + [Implementation](https://2021.igem.org/Team%3ASJTang/Implementation)
  + [Education](https://2021.igem.org/Team%3ASJTang/Education)
  + [Communication](https://2021.igem.org/Team%3ASJTang/Communication)
* Team
  + [About Us](https://2021.igem.org/Team%3ASJTang/Team)
  + [Attributions](https://2021.igem.org/Team%3ASJTang/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASJTang/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ASJTang/Partnership)
* Protocols
  + [Notebook](https://2021.igem.org/Team%3ASJTang/Notebook)
  + [Methods](https://2021.igem.org/Team%3ASJTang/Methods)
* [Awards](https://2021.igem.org/Team%3ASJTang/Awards)

##### Background

* [Market Study](https://2021.igem.org/Team%3ASJTang/Market_Study)
* [Policies](https://2021.igem.org/Team%3ASJTang/Policies)
* [Description](https://2021.igem.org/Team%3ASJTang/Description)

##### HP

* [Overview](https://2021.igem.org/Team%3ASJTang/Human_Practices)
* [Implementation](https://2021.igem.org/Team%3ASJTang/Implementation)
* [Education](https://2021.igem.org/Team%3ASJTang/Education)
* [Communication](https://2021.igem.org/Team%3ASJTang/Communication)

##### Project

* [Design](https://2021.igem.org/Team%3ASJTang/Design)
* [Results](https://2021.igem.org/Team%3ASJTang/Results)
* [Engineering](https://2021.igem.org/Team%3ASJTang/Engineering)
* [Contribution](https://2021.igem.org/Team%3ASJTang/Contribution)
* [Model](https://2021.igem.org/Team%3ASJTang/Model)
* [Proof of Concept](https://2021.igem.org/Team%3ASJTang/Proof_Of_Concept)
* [SDGs](https://2021.igem.org/Team%3ASJTang/Sustainable)
* [Safety](https://2021.igem.org/Team%3ASJTang/Safety)
* [Graphic Design](https://2021.igem.org/Team%3ASJTang/Graphic_Design)

##### Hardware

* Overview
* Machinery
* Software
* Interview

##### Protocols

* Notebook
* Methods

##### Team

* [About Us](https://2021.igem.org/Team%3ASJTang/Team)
* [Attributions](https://2021.igem.org/Team%3ASJTang/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ASJTang/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ASJTang/Partnership)

##### Awards

* ![](https://static.igem.org/mediawiki/2021/2/2f/T--SJTang--Footer_Bluepha.png)
* ![](https://static.igem.org/mediawiki/2021/f/f9/T--SJTang--Footer_SnapGene.png)
* [![](https://static.igem.org/mediawiki/2021/4/45/T--SJTang--Footer_WeChat.png)](https://2021.igem.org/Team%3ASJTang/Human_Practices)
* ![](https://static.igem.org/mediawiki/2021/5/54/T--SJTang--Footer_Email.png)

![](https://static.igem.org/mediawiki/2021/d/d4/T--SJTang--footerlog.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SJTang/Engineering

Team:SJTang - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/1/1a/T--SJTang--nav_icon.svg)](https://2021.igem.org/Team%3ASJTang)

* Background
  + [Description](https://2021.igem.org/Team%3ASJTang/Description)
  + ---
  + [Market Study](https://2021.igem.org/Team%3ASJTang/Market_Study)
  + [Policies](https://2021.igem.org/Team%3ASJTang/Policies)
* Project
  + [Design](https://2021.igem.org/Team%3ASJTang/Design)
  + [Results](https://2021.igem.org/Team%3ASJTang/Results)
  + [Engineering](https://2021.igem.org/Team%3ASJTang/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ASJTang/Contribution)
  + [Model](https://2021.igem.org/Team%3ASJTang/Model)
  + [Proof of Concept](https://2021.igem.org/Team%3ASJTang/Proof_Of_Concept)
  + [SDGs](https://2021.igem.org/Team%3ASJTang/Sustainable)
  + [Safety](https://2021.igem.org/Team%3ASJTang/Safety)
  + [Graphic Design](https://2021.igem.org/Team%3ASJTang/Graphic_Design)
* Hardware
  + [Overview](https://2021.igem.org/Team%3ASJTang/Hardware)
  + ---
  + [Machinery](https://2021.igem.org/Team%3ASJTang/Machinery)
  + [Software](https://2021.igem.org/Team%3ASJTang/Software)
  + [Interview](https://2021.igem.org/Team%3ASJTang/Hardware_Interview)
* Human Practice
  + [Overview](https://2021.igem.org/Team%3ASJTang/Human_Practices)
  + ---
  + [Implementation](https://2021.igem.org/Team%3ASJTang/Implementation)
  + [Education](https://2021.igem.org/Team%3ASJTang/Education)
  + [Communication](https://2021.igem.org/Team%3ASJTang/Communication)
* Team
  + [About Us](https://2021.igem.org/Team%3ASJTang/Team)
  + [Attributions](https://2021.igem.org/Team%3ASJTang/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASJTang/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ASJTang/Partnership)
* Protocols
  + [Notebook](https://2021.igem.org/Team%3ASJTang/Notebook)
  + [Methods](https://2021.igem.org/Team%3ASJTang/Methods)
* [Awards](https://2021.igem.org/Team%3ASJTang/Awards)

![](https://static.igem.org/mediawiki/2021/5/51/T--SJTang--Engineering_Cover.png)

## Engineering Success

Cyanobacteria are prokaryotic organisms that can be used for photosynthesis, depending on their characteristics of hydrogen produced by nitrogen fixation, and in order to further improve the hydrogen production efficiency of E. coli, we chose a membrane-binding hydrogenation enzyme Oxygen-tolerant [NiFe]-hydrogenase from Hydrogenovibrio marinus MH-110. The structure of this hydrogenase is similar to that in Escherichia coli, and it is also composed of two subunits. Among them, HoxG1(NCBI Locus: WP\_029908155) is the large subunit of hydrogenase, and HoxK1 (NCBI Locus: WP\_029908158) is the small subunit. The structure information of the two of them on NCBI is as follows:

![](https://static.igem.org/mediawiki/2021/6/66/T--SJTang--Engineering_img1.png)

![](https://static.igem.org/mediawiki/2021/3/33/T--SJTang--Engineering_img2.png)

Figure.1 Conserved domain of Hydrogenase. Up: HoxG1. Down: HoxK1

Furthermore, in the process of reviewing papers, we found that the first 132 bp of the HyaA and HoxK1 sequences both contain the signal sequence. In order for this protein to be successfully expressed heterologously in E. coli, we replaced the signal sequence of HoxK1 and replaced it with the signal sequence of HyaA. Finally, we have the following plasmid design:

![](https://static.igem.org/mediawiki/2021/0/05/T--SJTang--Engineering_img3.png)

Figure.2Plasmid design of Hydrogenase production plasmid.

Then we transformed the obtained plasmid into E. coli BL21. Cultivate the seed solution overnight, and then inoculate it into 100 mL LB liquid medium at a ratio of 1:50. Two groups start induction at 0h with 1mM IPTG. After culturing for 8 hours at 37 °C 220rpm, 1 mM IPTG is added for overnight induction at 18 °C. Afterwards, the bacteria were collected and disrupted. Since Hydrogenase is a membrane protein, the cell disruption suspension and the centrifuged supernatant were collected separately for protein SDS-PAGE electrophoresis. The results of electrophoresis are as follows:

![](https://static.igem.org/mediawiki/2021/4/41/T--SJTang--Engineering_img4.png)

Figure.3The SDS-PAGE result of cell disruption.

The results of electrophoresis showed that the expression of hydrogenase was successful and basically consistent with expectations. The results showed that the induction effect was better from 0h than 8h, and the content of HoxK1 and HoxG1 in the suspension was higher than that in the supernatant after centrifuge.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SJTang/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

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SJTang/Results

Team:SJTang - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/1/1a/T--SJTang--nav_icon.svg)](https://2021.igem.org/Team%3ASJTang)

* Background
  + [Description](https://2021.igem.org/Team%3ASJTang/Description)
  + ---
  + [Market Study](https://2021.igem.org/Team%3ASJTang/Market_Study)
  + [Policies](https://2021.igem.org/Team%3ASJTang/Policies)
* Project
  + [Design](https://2021.igem.org/Team%3ASJTang/Design)
  + [Results](https://2021.igem.org/Team%3ASJTang/Results)
  + [Engineering](https://2021.igem.org/Team%3ASJTang/Engineering)
  + [Contribution](https://2021.igem.org/Team%3ASJTang/Contribution)
  + [Model](https://2021.igem.org/Team%3ASJTang/Model)
  + [Proof of Concept](https://2021.igem.org/Team%3ASJTang/Proof_Of_Concept)
  + [SDGs](https://2021.igem.org/Team%3ASJTang/Sustainable)
  + [Safety](https://2021.igem.org/Team%3ASJTang/Safety)
  + [Graphic Design](https://2021.igem.org/Team%3ASJTang/Graphic_Design)
* Hardware
  + [Overview](https://2021.igem.org/Team%3ASJTang/Hardware)
  + ---
  + [Machinery](https://2021.igem.org/Team%3ASJTang/Machinery)
  + [Software](https://2021.igem.org/Team%3ASJTang/Software)
  + [Interview](https://2021.igem.org/Team%3ASJTang/Hardware_Interview)
* Human Practice
  + [Overview](https://2021.igem.org/Team%3ASJTang/Human_Practices)
  + ---
  + [Implementation](https://2021.igem.org/Team%3ASJTang/Implementation)
  + [Education](https://2021.igem.org/Team%3ASJTang/Education)
  + [Communication](https://2021.igem.org/Team%3ASJTang/Communication)
* Team
  + [About Us](https://2021.igem.org/Team%3ASJTang/Team)
  + [Attributions](https://2021.igem.org/Team%3ASJTang/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASJTang/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ASJTang/Partnership)
* Protocols
  + [Notebook](https://2021.igem.org/Team%3ASJTang/Notebook)
  + [Methods](https://2021.igem.org/Team%3ASJTang/Methods)
* [Awards](https://2021.igem.org/Team%3ASJTang/Awards)

![](https://static.igem.org/mediawiki/2021/c/c8/T--SJTang--Results_Cover.png)

## Overview

Overall, we successfully synthesized I-HTCC and achieved [Ni-Fe] Hydrogenase expression in E. coli. In the part of Rhodopseudomonas palustris, we conducted various investigations on the newly introduced iGEM chassis bacteria and tried to knock out genes. However, in the end, due to some unknown reasons, we were not able to successfully achieve hydrogen synthesis using modified Escherichia coli or Rhodopseudomonas palustris.

## 1. E. coli Hydrogen Production

#### 1.1 I-HTCC Production

We prepared the I-HTCC material according to the method in the reference. After hydrothermal reaction, filtration, vacuum drying and grinding, the finished products are as follows:

![](https://static.igem.org/mediawiki/2021/d/dc/T--SJTang--Results1.png)

Figure.1I-HTCC finished product. Left: the powder obtained after grinding; Right: IHTCC finished product stored in deionized water.

#### 1.2 Hydrogenase Production

According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows:

![](https://static.igem.org/mediawiki/2021/f/f1/T--SJTang--Results2.png)

Figure.2Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%).

Both HoxG1 (Hydrogenase Large subunit) and HoxK1 (Hydrogenase small subunit) are from Hydrogenovibrio marinus MH-110. The signal sequence of HoxK1 is replaced with HyaA (Hydrogenase small subunit) signal sequence from E. coli BL21. The agarose gel shows the length of the plasmid is right.

Then we transformed the obtained plasmid into E. coli BL21. Cultivate the seed solution overnight, and then inoculate it into 100 mL LB liquid medium at a ratio of 1:50. Two groups start induction at 0h with 1mM IPTG. After culturing for 8 hours at 37 °C 220rpm, 1 mM IPTG is added for overnight induction at 18 °C. Afterwards, the bacteria were collected and disrupted. Since Hydrogenase is a membrane protein, the cell disruption suspension and the centrifuged supernatant were collected separately for protein SDS-PAGE electrophoresis. The results of electrophoresis are as follows:

![](https://static.igem.org/mediawiki/2021/f/f7/T--SJTang--Results3.png)

Figure.3The SDS-PAGE result of cell disruption.

The results of electrophoresis showed that the expression of hydrogenase was successful and basically consistent with expectations. The results showed that the induction effect was better from 0h than 8h, and the content of HoxK1 and HoxG1 in the suspension was higher than that in the supernatant after centrifuge.

#### 1.3 Hydrogen Production

After completing the preparation of IHTCC and the induced expression of hydrogenase, we tried to test the combination of the two. Since I-HTCC is negatively charged, after using PAH(Polyallylamine hydrochloride) to make the surface of E. coli positively charged, the two are mixed. The status of the bacteria obtained after mixing is as follows:

![](https://static.igem.org/mediawiki/2021/9/9a/T--SJTang--Results4.png)

Figure.4I-HTCC adding to E. coli. Left: I-HTCC@E. coli, right: I-HTCC with E. coli.

Since I-HTCC is negatively charged and the PAH treated E. coli is positively charged, the two can bind to each other under the action of electrostatic force (I-HTCC@E. coli). The untreated Escherichia coli cannot combine well with I-HTCC, and the two will have an obvious layered state after centrifugation (I-HTCC with E. coli). The results in the above figure show that the E. coli we processed can bind well to the I-HTCC we prepared.

After that, we used the processed E. coli to conduct a gas production fermentation experiment. However, the results showed that no hydrogen was produced.

## 2. R. palustris Hydrogen Production

#### 2.1 Gene Knock-out for R. palustris

In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows:

![](https://static.igem.org/mediawiki/2021/f/fe/T--SJTang--Results5.png)

Figure.5Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).

After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows:

![](https://static.igem.org/mediawiki/2021/5/5c/T--SJTang--Results6.png)

Figure.6Gene knockout design and result. Left: Test Design. Right:HupS Test Result.

Due to the time of the project, we completed the knockout of HupS, but the knockout screening of UppE was not successful. In the end, we failed to complete the knockout of UppE.

#### 2.2 Hydrogen Production with R. palustris

We cultivated Rhodopseudomonas palustris to a certain concentration and then inoculate it into a culture flask for airtight fermentation. However, due to some reasons that we don't know, no hydrogen yield was observed in the end.

![](https://static.igem.org/mediawiki/2021/c/c5/T--SJTang--Results7.png)

Figure.7R. palustris in YPMOPS medium.

![](https://static.igem.org/mediawiki/2021/c/c3/T--SJTang--Results8.png)

Figure.8R. palustris with our Hardware.

##### Background

* [Market Study](https://2021.igem.org/Team%3ASJTang/Market_Study)
* [Policies](https://2021.igem.org/Team%3ASJTang/Policies)
* [Description](https://2021.igem.org/Team%3ASJTang/Description)

##### HP

* [Overview](https://2021.igem.org/Team%3ASJTang/Human_Practices)
* [Implementation](https://2021.igem.org/Team%3ASJTang/Implementation)
* [Education](https://2021.igem.org/Team%3ASJTang/Education)
* [Communication](https://2021.igem.org/Team%3ASJTang/Communication)

##### Project

* [Design](https://2021.igem.org/Team%3ASJTang/Design)
* [Results](https://2021.igem.org/Team%3ASJTang/Results)
* [Engineering](https://2021.igem.org/Team%3ASJTang/Engineering)
* [Contribution](https://2021.igem.org/Team%3ASJTang/Contribution)
* [Model](https://2021.igem.org/Team%3ASJTang/Model)
* [Proof of Concept](https://2021.igem.org/Team%3ASJTang/Proof_Of_Concept)
* [SDGs](https://2021.igem.org/Team%3ASJTang/Sustainable)
* [Safety](https://2021.igem.org/Team%3ASJTang/Safety)
* [Graphic Design](https://2021.igem.org/Team%3ASJTang/Graphic_Design)

##### Hardware

* Overview
* Machinery
* Software
* Interview

##### Protocols

* Notebook
* Methods

##### Team

* [About Us](https://2021.igem.org/Team%3ASJTang/Team)
* [Attributions](https://2021.igem.org/Team%3ASJTang/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ASJTang/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ASJTang/Partnership)

##### Awards

* ![](https://static.igem.org/mediawiki/2021/2/2f/T--SJTang--Footer_Bluepha.png)
* ![](https://static.igem.org/mediawiki/2021/f/f9/T--SJTang--Footer_SnapGene.png)
* [![](https://static.igem.org/mediawiki/2021/4/45/T--SJTang--Footer_WeChat.png)](https://2021.igem.org/Team%3ASJTang/Human_Practices)
* ![](https://static.igem.org/mediawiki/2021/5/54/T--SJTang--Footer_Email.png)

![](https://static.igem.org/mediawiki/2021/d/d4/T--SJTang--footerlog.png)