iGEM 2018 — Team HUST-China

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# Team:HUST-China/Notebook

iGEM 2018 HUST-China

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* [Home](https://2018.igem.org/Team%3AHUST-China)
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* Model
  + [Modeling overview](https://2018.igem.org/Team%3AHUST-China/Modeling%20overview)
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  + [Software](https://2018.igem.org/Team%3AHUST-China/Software)
* PARTS
  + [Parts Overview](https://2018.igem.org/Team%3AHUST-China/Parts%20Overview)
  + [Composite part](https://2018.igem.org/Team%3AHUST-China/Composite%20part)
  + [Part collection](https://2018.igem.org/Team%3AHUST-China/Part%20collection)
* HP
  + [Human Practices](https://2018.igem.org/Team%3AHUST-China/Human%20Practices)
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* TEAM
  + [Team Members](https://2018.igem.org/Team%3AHUST-China/Team)
  + [Collaborations](https://2018.igem.org/Team%3AHUST-China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AHUST-China/Attributions)

## NoteBook

* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*April 1-30

  Ziyang Xiao, Junhao Xiong, Jingyu Ren, Yan chen, Qian Li
  Pre-experiment of electrogenesis: tested and optimized the electrogenesis condition for MFC (Microbial Fuel Cell)
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*May 1-31

  Yiyan Yu, Ziyi Li, Junhao Xiong
  Basic design of molecular experiments: design sequences, primers and the construction of Shewanella
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*July 1-7

  Junhao Xiong, Ziqi Yin, Jiayi Liu
  Preparation for following experiments including preparing LB culture medium, antibiotics and optimizing culture condition of Shewanella oneidensis MR-1.

  Bo Peng
  Construction of pSB1C3-RBS-mleS

  ![](https://static.igem.org/mediawiki/2018/7/77/T--HUST-China--2018-notebook01.png)

  Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu
  synechocystis Group took the responsibility of doing the Interlab. Preparation for the experiment.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*July 8-21

  Junhao Xiong, Ziqi Yin, Jiayi Liu
  Construction of pYYDT-pflB: PCR, double enzyme digestion (with NdeI & SalI), ligation, transformation (into E.coli TOP10).

  ![](https://static.igem.org/mediawiki/2018/1/12/T--HUST-China--2018-notebook02.png)

  Qian Li
  Construction of pSB1C3-RBS-lldp-RBS-ldhA-TT

  ![](https://static.igem.org/mediawiki/2018/2/2b/T--HUST-China--2018-notebook03.png)

  Ziyang Xiao, Haotian Ren, Yan Chen, Hao Qiu
  Finishing the cell cultures cultivation and using some machines to measure some related data.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*July 22-29

  Junhao Xiong, Ziqi Yin, Jiayi Liu
  Construction of pYYDT-gapA-mdh, pYYDT-lldEFG: PCR, double enzyme digestion (with NdeI & SalI), ligation, transformation (into E.coli TOP10).

  ![](https://static.igem.org/mediawiki/2018/3/37/T--HUST-China--2018-notebook04.png)

  Haibo Huang
  Construction of pSB1C3-RBS-lldP and pSB1C3-RBS-ldhA.

  ![](https://static.igem.org/mediawiki/2018/a/a3/T--HUST-China--2018-notebook05.png)

  Haotian Ren
  Restriction enzyme digestion of psb1c3
  Ziyang Xiao, Yan Chen, Haotian Ren
  Testing the cultivation ways of synechocystis PCC6803
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*July 30 - August 5

  Ziyi Li, Jiayi Liu, Yizhe Zeng
  Construction of pYYDT-gapA, pYYDT-mdh
  Standardization of gapA, mdh

  ![](https://static.igem.org/mediawiki/2018/e/e6/T--HUST-China--2018-notebook06.png)

  ![](https://static.igem.org/mediawiki/2018/0/08/T--HUST-China--2018-notebook07.png)

  ![](https://static.igem.org/mediawiki/2018/8/8c/T--HUST-China--2018-notebook08.png)

  Kaiwen Liu
  Construction of pmg105-PpckA-RBS-lldP-RBS-ldhA-TT

  ![](https://static.igem.org/mediawiki/2018/4/4a/T--HUST-China--2018-notebook09.png)

  Yan Chen, Sijie Xu
  Interlab Experiment 4
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*August 6-12

  Ziyi Li, Jiayi Liu, Yizhe Zeng
  Construction of pYYDT-dld
  Standardization of dld, lldEFG

  ![](https://static.igem.org/mediawiki/2018/2/20/T--HUST-China--2018-notebook10.png)

  Haibo Huang
  Construction of pSB1C3-RBS-ldhA-RBS-lldP-TT

  ![](https://static.igem.org/mediawiki/2018/b/b2/T--HUST-China--2018-notebook11.png)

  Yan Chen, Yuanda Huang
  Restriction enzyme digestion of psb-lldEFG
  Yan Chen, Sijie Xu, Yuanda Huang, Ziyang Xiao
  Constructing ldhDc-lldP, ldhDARSDR-lldP, ldhDnARSDR-lldPT
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*August 13-19

  Ziyi Li, Yizhe Zeng
  Overlap extension PCR of dld and lldEFG: We got the right band but the sequencing result revealed a mutation of it.

  ![](https://static.igem.org/mediawiki/2018/9/91/T--HUST-China--2018-notebook12.png)

  Making competent cells of E.coli WM3064: we need the deficiency type WM3064 to conjugate with Shewanella oneidensis MR-1.
  Bo Peng
  Construction of pmg105-PpckA-RBS-lldP-TT, pmg105-PpckA-RBS-ldhA-RBS-lldP-TT and pmg105-PpckA-RBS-mleS-TT.

  ![](https://static.igem.org/mediawiki/2018/0/0e/T--HUST-China--2018-notebook13.png)

  Yuanda Huang, Yan Chen, Sijie Xu, Tao Jian, Ziyang Xiao
  PCR of ldhDnARSdR, PCR of ldhDARSdR,
  Overlap extension PCR of ldhDnARSdR and lldp (Dn-L)
  Overlap extension PCR of ldhDARSdR and lldp (DA-L)
  Overlap extension PCR of ldhDC and lldp (DC-L)
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*August 20-26

  Ziyi Li, Junhao Xiong, Yizhe Zeng
  Conjugation of E.coli WM3064 and Shewanella oneidensis MR-1: we transferred the targeted plasmid into E.coli WM3064 and conjugated WM3064 and Shewanella oneidensis MR-1 so that the Shewanella oneidensis MR-1 could finally get the targetted plasmid.
  Conjugation pYYDT and pYYDT-lldEFG this week.

  ![](https://static.igem.org/mediawiki/2018/f/f4/T--HUST-China--2018-notebook14.png)

  Using device to produce electricity with Shewanella. We proved that Shewanella could produce more electricity with the existence of lactate.

  ![](https://static.igem.org/mediawiki/2018/4/4a/T--HUST-China--2018-notebook15.png)

  Yuanda Huang, Haotian Ren, Ziyang Xiao
  PCR of ldhDnARSdR, PCR of ldhDARSdR,
  PCR of ldhDnARSdR, extraction enzyme digestion of ldhDnARSdR,
  Ligation of ldhDnARSdR and psb1c3, transformation
  Hao Qiu
  Constructing the pathway of TH-glad-lldp.
  Insert sequence TH-glad-lldp to vector psb1c3.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*August 27 – September 2

  Ziyi Li, Junhao Xiong, Yizhe Zeng
  Redesigning about connecting dld and lldEFG: We decided to drop the idea about overlap extension PCR to connect dld and lldEFG and started to use isocaudamer (XbaI and SpeI) to connect these genes.

  ![](https://static.igem.org/mediawiki/2018/7/7a/T--HUST-China--2018-notebook16.png)

  Conjugation of pYYDT-gapA and pYYDT-mdh.
  By constructing Rhodopseudomonas palustris-Shewanella oneidensis mutualism system, we found that Shewanella could produce more electricity if Rhodopseudomonas palustris exists.
  Qian Li
  Construction of pSB1C3-RBS-mleS-RBS-lldp.

  ![](https://static.igem.org/mediawiki/2018/c/c0/T--HUST-China--2018-notebook17.png)

  Yuanda Huang, Ziyang Xiao
  Overlap extension PCR of ldhDnARSdR-lldp

  ![](https://static.igem.org/mediawiki/2018/e/e2/T--HUST-China--2018-notebook18.png)

  Tao Jian
  Adding Flag tag to ldhD-lldP.
  Yan Chen
  Adding Flag tag to ldhDc-lldP
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*September 3-9

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  Conjugation of pYYDT-dld and pYYDT-Ptac-pflB.

  ![](https://static.igem.org/mediawiki/2018/1/1a/T--HUST-China--2018-notebook19.png)

  New method to connect dld and lldEFG. However, we failed at the construction of pSB1C3-ptac-lldEFG because of the biobrick prefix on pYYDT.
  We proved that Shewanella could produce more electricty under anaerobic condition.
  Yuanda Huang
  Adding tags to ldhDnARSdR-lldp
  Yan Chen
  Adding his tag to ldhD-lldP and ldhDc-lldP
  Hao Qiu
  Amplifying the gene and transforming in E. coli.
  Ziyang Xiao
  synechocystis culture.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*September 10-16

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  Conjugation of pYYDT-gapA-mdh

  ![](https://static.igem.org/mediawiki/2018/9/9f/T--HUST-China--2018-notebook20.png)

  Haibo Huang
  Adding tags to ldhDnARSdR-lldp
  Yan Chen
  Construction of pmg105-PpckA-RBS-ldhA-TT.

  ![](https://static.igem.org/mediawiki/2018/e/e2/T--HUST-China--2018-notebook21.png)

  Yuanda Huang
  Adding tags to ldhDARSdR-lldp
  Yan Chen
  Recombination of ldhD-lldP and ldhDc-lldP to shuttle plasmid PCK306
  Hao Qiu
  Checking the finial consequence by gene sequencing.
  Ziyang Xiao
  synechocystis culture.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*September 17-23

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  Application of the electrogenesis: we used the device to charge an LED (1.7V), a cell phone (5V) and a small engine (5V). The success proved that we can put our device into practice.
  We proved that carbon cloth has better effect than carbon rods.
  Kaiwen Liu
  Construction of pmg105-PpckA-RBS-mleS-RBS-lldP-TT

  ![](https://static.igem.org/mediawiki/2018/3/39/T--HUST-China--2018-notebook22.png)

  Haibo Huang
  Inserting ldhDnARSdR-lldp biaoda into the plasmid called pck306 through recombinase,
  Inserting ldhDARSdR-lldp biaoda into the plasmid called pck306 through recombinase

  ![](https://static.igem.org/mediawiki/2018/1/1f/T--HUST-China--2018-notebook23.png)

  Yan Chen
  Transforming recombinant shuttle plasmid into synechocystis
  Hao Qiu
  Transforming ldhd sequence into synechocystis.
  Ziyang Xiao
  synechocystis culture.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*September 24-30

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  Construction of pSB1C3-pflB-fdh:we found that there was no tac promotor before.

  ![](https://static.igem.org/mediawiki/2018/b/bd/T--HUST-China--2018-notebook24.png)

  We proved that the electricity produced by Rhodopseudomonas palustrls could be ignored in the electrogenesis system.
  At the same time, it was demonstrated that the lactate could be the only carbon source in the culture medium.

  Qian Li
  Construction of pSB1C3- RBS-mleS-RBS-lldP-RBS-ldhA-TT

  ![](https://static.igem.org/mediawiki/2018/1/1a/T--HUST-China--2018-notebook25.png)

  Hao Qiu
  Using DNS to detect the condition of producing lactate by synechocystis.
  Yuanda Huang, Yan Chen
  Transforming recombinant shuttle plasmid into synechocystis.
  Ziyang Xiao
  synechocystis culture.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*October 1-7

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  Construction of pYYDT-pflB-fdh

  ![](https://static.igem.org/mediawiki/2018/5/5c/T--HUST-China--2018-notebook26.png)

  Conjugation of pYYDT-pflB-fdh
  We proved that the engineering bacteria with pYYDT-gapA-mdh could produce more electricity than the bacteria with pYYDT.
  Bo Peng
  Construction of RBS-mleS-RBS-lldP-TT pmg105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-tt.

  ![](https://static.igem.org/mediawiki/2018/5/57/T--HUST-China--2018-notebook27.png)

  Yuanda Huang
  Overlap extension PCR of ldhDnARSdR and lldp.
  Hao Qiu
  Expansion culture of synechocystis. DNS detection of sugar production of synechocystis, YFP fluorescent protein detection of synechocystis.
  Yan Chen
  Transforming recombinant shuttle plasmid into synechocystis.
* *![](https://static.igem.org/mediawiki/2018/8/89/T--HUST-China--2018-coin04.png)*October 8-14

  Ziyi Li, Junhao Xiong, Yizhe Zeng, Ziqi Yin
  1. Construction of pSB1C3-Ptac-pflB-fdh
  2. Reconjugation of pYYDT-lldEFG
  3. We did two groups of electrogenesis experiments. One of them included the engineering synechocystis and Shewanella, the other included the engineering Rhodopseudomonas palustris and Shewanella. It turned out that the later one worked better than the former one.
  Yuanda Huang
  Add the first tag to ldhDnARSdR-lldp /ldhDARSdR-lldp.
  Hao Qiu
  Detection of YFP fluorescent protein of synechocystis.
  Yan Chen
  Detection of YFP fluorescent protein of synechocystis. Detection of Flag tag.
  Ziyang Xiao
  Detection of YFP fluorescent protein of synechocystis. Detection of 6× His tag and lactate.
  Conjugation of pYYDT-pflB-fdh
  We proved that the engineering bacteria with pYYDT-gapA-mdh could produce more electricity than the bacteria with pYYDT.
  Yuanda Huang
  Overlap extension PCR of ldhDnARSdR and lldp.
  Hao Qiu
  Expansion culture of synechocystis. DNS detection of sugar production of synechocystis, YFP fluorescent protein detection of synechocystis.
  Yan Chen
  Transforming recombinant shuttle plasmid into synechocystis.

College of Life Science & Technology
Huazhong University of Science and Technology
Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C: 430074
Copyright ©2018 Huazhong University of Science & Technology. Produced By [HUST-China](https://2018.igem.org/Team%3AHUST-China)

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# Team:HUST-China/Experiments

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* [Home](https://2018.igem.org/Team%3AHUST-China)
* PROJECT
  + [Background](https://2018.igem.org/Team%3AHUST-China/project/background)
  + [Description](https://2018.igem.org/Team%3AHUST-China/Description)
  + [Applied Design](https://2018.igem.org/Team%3AHUST-China/Applied%20Design)
  + [Safety](https://2018.igem.org/Team%3AHUST-China/Safety)
  + [Demonstrate](https://2018.igem.org/Team%3AHUST-China/Demonstrate)
* WETLAB
  + [Experiments](https://2018.igem.org/Team%3AHUST-China/Experiments)
  + [Results](https://2018.igem.org/Team%3AHUST-China/Results)
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* HP
  + [Human Practices](https://2018.igem.org/Team%3AHUST-China/Human%20Practices)
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* TEAM
  + [Team Members](https://2018.igem.org/Team%3AHUST-China/Team)
  + [Collaborations](https://2018.igem.org/Team%3AHUST-China/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3AHUST-China/Attributions)

## Experiments

### **Part1: Photosynthetic microorganism system**

*![](https://static.igem.org/mediawiki/2018/8/8d/T--HUST-China--2018-lanzao01.png)*

### **1.1 Synechocystis sp.**

### **1.1.1 Plasmid Construction in E.coli**

We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-TH-RBS-gldA-RBS-lldp | pSB1C3 | E.coli DH5α |

![](https://static.igem.org/mediawiki/2018/b/bf/T--HUST-China--2018-experiment-picture12.png.png)

### **1.1.2 Add Flag Sequence into parts**

The constructed plasmid was transformed into E.coli Top 10.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |

### **1.1.3 Add 6×His Sequence into parts**

The constructed plasmid was transformed into E.coli Top 10.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |

### **1.1.4 pCK360 Plasmid Conjugation**

We constructed following vectors carrying our parts and transformed these vectors into E.coli Top 10 to transformed into cynobacteria.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-ldhD-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDC-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDnARSdR-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDARSdR-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |

![](https://static.igem.org/mediawiki/2018/f/f0/T--HUST-China--2018-experimental_6-ldhD-flag-lldP-his.png)

Figure 6. modified circuit of ldhD-lldP.

![](https://static.igem.org/mediawiki/2018/a/ae/T--HUST-China--2018-experimental_7-ldhDC-flag-lldP-his.png)

Figure 7. modified circuit of ldhDC-lldP.

![](https://static.igem.org/mediawiki/2018/8/8e/T--HUST-China--2018-experimental_8-ldhDnARSdR-flag-lldP-his.png)

Figure 8. modified circuit of ldhDnARSdR-lldP.

![](https://static.igem.org/mediawiki/2018/2/27/T--HUST-China--2018-experimental_9-ldhDARSdR-flag-lldP-his.png)

Figure 9. modified circuit of ldhDARSdR-lldP.

### **1.1.5 Detection of yellow fluorescent protein**

Since plasmid pCK306 has yellow fluorescent protein sequence, we cultivated the engineering cynobacteria induced by rhamnose for 24h. And put them under the fluorescence microscope at the wave of 488nm excitation light

*![](https://static.igem.org/mediawiki/2018/6/63/T--HUST-China--2018-zhaozehong01.png)*

### ****1.2 Rhodopseudomonas palustris****

### **1.2.1 Plasmid Construction in E.coli**

We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.

![](https://static.igem.org/mediawiki/2018/3/3e/T--HUST-China--2018-description-picture20.png.png)

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-mleS | pSB1C3 | E.coli TOP10 |
| RBS-ldhA | pSB1C3 | E.coli TOP10 |
| RBS-lldP | pSB1C3 | E.coli TOP10 |
| RBS-mleS-RBS-lldP | pSB1C3 | E.coli TOP10 |
| RBS-lldP-RBS-ldhA-TT | pSB1C3 | E.coli TOP10 |
| RBS-ldhA-RBS-lldP-TT | pSB1C3 | E.coli TOP10 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pSB1C3 | E.coli TOP10 |
| RBS-mleS | pMG105 | E.coli TOP10 |
| RBS-ldhA | pMG105 | E.coli TOP10 |
| RBS-lldP | pMG105 | E.coli TOP10 |
| RBS-mleS-RBS-lldP | pMG105 | E.coli TOP10 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | E.coli TOP10 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | E.coli TOP10 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | E.coli TOP10 |

### **1.2.2 Transformation into E.coli BL21**

The constructed plasmid was transformed into E.coli BL21.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-mleS | pMG105 | E.coli BL21 |
| RBS-ldhA | pMG105 | E.coli BL21 |
| RBS-lldP | pMG105 | E.coli BL21 |
| RBS-mleS-RBS-lldP | pMG105 | E.coli BL21 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | E.coli BL21 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | E.coli BL21 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | E.coli BL21 |

### **1.2.3 Transformation into Rhodopseudomonas palustris DSM5859**

The constructed plasmid was transformed into Rhodopseudomonas palustris DSM5859.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-mleS | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-ldhA | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-lldP | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-mleS-RBS-lldP | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |

### **1.2.4 Verification the expression at mRNA level**

We did RT-qPCR to test the relative expression levels of each genes.

### **1.2.5 Detection of Lactate**

We detect lactate by Lactic Acid assay kit.

### **Part2: Electrogenic microorganism system:**

*![](https://static.igem.org/mediawiki/2018/0/0c/T--HUST-China--2018-xiwa01.png)*

### **Shewanella oneidensis MR-1**

### **2.1 Plasmid Construction in E.coli**

We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.

![](https://static.igem.org/mediawiki/2018/b/bd/T--HUST-China--2018-descripion-picture13.png.png)

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-dld | pSB1C3 | E.coli TOP10 |
| RBS-lldEFG | pSB1C3 | E.coli TOP10 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pSB1C3 | E.coli TOP10 |
| RBS-gapA | pSB1C3 | E.coli TOP10 |
| RBS-mdh | pSB1C3 | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT | pSB1C3 | E.coli TOP10 |
| RBS-Ptac -RBS-pflB | pSB1C3 | E.coli TOP10 |
| RBS-fdh | pSB1C3 | E.coli TOP10 |
| RBS-pflB-RBS-fdh | pSB1C3 | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pSB1C3 | E.coli TOP10 |
| RBS-dld | pYYDT | E.coli TOP10 |
| RBS-lldEFG | pYYDT | E.coli TOP10 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | E.coli TOP10 |
| RBS-gapA | pYYDT | E.coli TOP10 |
| RBS-mdh | pYYDT | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT | pYYDT | E.coli TOP10 |
| RBS-Ptac -RBS-pflB | pYYDT | E.coli TOP10 |
| RBS-pflB-RBS-fdh | pYYDT | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | E.coli TOP10 |

### **2.2 Transformation into E.coli WM3064**

The constructed plasmid was transformed into a 2,6-diaminopimelic acid deficiency type E.coli WM3064.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-dld | pYYDT | E .coli WM3064 |
| RBS-lldEFG | pYYDT | E .coli WM3064 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | E .coli WM3064 |
| RBS-gapA | pYYDT | E .coli WM3064 |
| RBS-mdh | pYYDT | E .coli WM3064 |
| RBS-gapA-RBS-mdh-TT | pYYDT | E .coli WM3064 |
| RBS-Ptac -RBS-pflB | pYYDT | E .coli WM3064 |
| RBS-pflB-RBS-fdh | pYYDT | E .coli WM3064 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | E .coli WM3064 |

### **2.3 Conjugation between E.coli and Shewanella oneidensis MR-1**

We did conjugation after the transformation to WM3064 to introduce the plasmid into Shewanella oneidensis MR-1.

| Part | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| RBS-dld | pYYDT | Shewanella oneidensis MR-1 |
| RBS-lldEFG | pYYDT | Shewanella oneidensis MR-1 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA | pYYDT | Shewanella oneidensis MR-1 |
| RBS-mdh | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA-RBS-mdh-TT | pYYDT | Shewanella oneidensis MR-1 |
| RBS-Ptac -RBS-pflB | pYYDT | Shewanella oneidensis MR-1 |
| RBS-pflB-RBS-fdh | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | Shewanella oneidensis MR-1 |

### **2.4 Verification the expression on mRNA level**

We did RT-qPCR to test the relative expression levels of each genes.

### **2.5 Electrogenesis detection**

We cultivated the engineering bacteria and put them into the electrogenesis device to test the output votage. We also did the measurement of electrode-attached biomass.

### **Co-culture and electricity**

| Microorganism 1 | Microorganism 2 | Addition 1 | Addition 2 |
| --- | --- | --- | --- |
| E.coli |  | LB medium |  |
| Shewanella oneidensis MR-1 |  | LB medium |  |
| Shewanella oneidensis MR-1 |  | LB medium | Lactate |
| Shewanella oneidensis MR-1 | Cynobacteria | LB medium |  |
| Shewanella oneidensis MR-1 | Rhodopseudomonas palustris DSM5859 | LB medium |  |
| Shewanella oneidensis MR-1（pYYDT- RBS-dld） |  | LB medium | Lactate |
| Shewanella oneidensis MR-1(pYYDT-RBS-lldEFG） |  | LB medium | Lactate |
| Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） |  | LB medium | Lactate |
| Shewanella oneidensis MR-1（pYYDT-RBS-gapA-RBS-mdh-TT ） |  | LB medium | Lactate |
| Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） |  | LB medium | Lactate |
| Shewanella oneidensis MR-1（pYYDT-RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB |  | LB medium | Lactate |
 Shewanella oneidensis MR-1（pYYDT- RBS-dld） | Rhodopseudomonas palustris DSM5859 | LB medium |  | Shewanella oneidensis MR-1（pYYDT-RBS-lldEFG） | Rhodopseudomonas palustris DSM5859 | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） | Rhodopseudomonas palustris DSM5859 | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT ） | Rhodopseudomonas palustris DSM5859 | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） | Rhodopseudomonas palustris DSM5859 | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB） | Rhodopseudomonas palustris DSM5859 | LB medium |  |
 Shewanella oneidensis MR-1（pYYDT- RBS-dld） | Cynobacteria | LB medium |  | Shewanella oneidensis MR-1（pYYDT-RBS-lldEFG） | Cynobacteria | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） | Cynobacteria | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT ） | Cynobacteria | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） | Cynobacteria | LB medium |  | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB） | Cynobacteria | LB medium |  |

### **Part3: Whole design**

*![](https://static.igem.org/mediawiki/2018/9/98/T--HUST-China--2018-coin17.png)*

In this part, we mainly verify our vision by constructing MFC system and we detect the generated voltage. We did several parallel experiments to prove it:

### **1. Pre-experiment of electrogenesis.**

MFC anode: Shewanella

MFC anode: Blank medium

MFC anode: E.coli

### **2. Lactate contrast.**

MFC anode: Shewanella

MFC anode: Shewanella+lactate

### **3. Demonstrate that carbon cloth has higher electricity production efficiency than carbon rod.**

MFC anode: carbon cloth

MFC anode: carbon rod

### **4. Study the effects of oxygen on Co-culture power systems.**

MFC anode: anaerobic

MFC anode: aerobic

### **5. Contrast the symbiotic effect of wild-type strains**

MFC anode: Synechocystis PCC6803 (wild type) + Shewanella

MFC anode: Rhodopseudomonas palustris (wild type) + Shewanella

### **6. Functional verification of engineered Rhodopseudomonas palustris**

MFC anode: Rhodopseudomonas palustris (wild type) + Shewanella

MFC anode: Rhodopseudomonas palustris(engineered type) + Shewanella

### **7. Functional verification of engineered Synechococcus PCC6803**

MFC anode: Synechocystis PCC6803 (wild type) + Shewanella

MFC anode: Synechocystis PCC6803 (engineered type) + Shewanella

College of Life Science & Technology
Huazhong University of Science and Technology
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## Results

### **Part1: Photosynthetic microorganism system**

*![](https://static.igem.org/mediawiki/2018/8/8d/T--HUST-China--2018-lanzao01.png)*

### **1.1 Synechocystis sp**

### **1.1.1 Verification of transformation**

pCK306 plasmid contains with yellow fluorescent protein. Therefore, to show pCK306 expresses successfully in Synechocystis sp. PCC 6803(cyanobacteria), we use fluorescence microscope to test whether it was transformed into bacteria or not. After cultivating for one week, we use 10μcyanobacteria cultivate dripping on the slides.

![](https://static.igem.org/mediawiki/2018/c/cf/T--HUST-China--2018-result-YFP.png)

Figure 1. Results of yellow fluorescent protein of pCK306 plasmid in Synechocystis PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.

As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.

### **1.1.2 Verification of gene expression**

**Affinity Chromatography**

In addition to knowing transcription of the gene, we want to exhibit the expression of the protein, we insert 6Xhis-tag into lldP and did affinity chromatography to show we finally made it. The result is shown below:

![](https://static.igem.org/mediawiki/2018/6/68/T--HUST-China--2018-result-lldP.png)

Figure 2a. There were nearly 0.2g bacteria used to testify the expression of lldP.
 After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis

Because of the slow growth of Synechocystis sp. PCC 6803 and time limitation, we did not have too much bacteria, so we only use nearly 0.2g of them to show whether lldP expressed. The color is pale but still can be seen. lldP expression succeeds.

**Real-Time Quantitative PCR**

To illustrate our project does work. We must prove that our circuit express successfully. Therefore, we did real-time quantitative PCR to demostrate the transcription of the circuit ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP and ldhDARSdR-lldP. The result shows as the figure below:

![](https://static.igem.org/mediawiki/2018/2/20/T--HUST-China--2018-result_xiaoziyang_hong.png)
![](https://static.igem.org/mediawiki/2018/a/ab/T--HUST-China--2018-result_xiaoziyang_zi.png)
![](https://static.igem.org/mediawiki/2018/9/96/T--HUST-China--2018-result_xiaoziyang_lan.png)
![](https://static.igem.org/mediawiki/2018/8/8d/T--HUST-China--2018-result_xiaoziyang_ju.png)

Figure 2b. The figure shows that ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP express successfully.

### **1.1.3 Detection of Lactate**

Finally, we should testify that the whole circuit in Synechocystis sp. PCC 6803(cyanobacteria) works. So we did lactate detection experiment. After adding L-Rhamose(1g/L) for 72h, we use Lactic Acid assay kit, provided by Nanjing Jiancheng Bioengineering, to quantify lactate concentration. The result is shown below, all the cyanobacteria cultivates are converting to OD750=1.

![](https://static.igem.org/mediawiki/2018/d/d6/T--HUST-China--2018-lactate_%281%29.png)

Figure 3.  *Lactate production of engineered Synechocystis sp. PCC6803.* Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days, then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration has shown above.

The figure indicates that after transforming our circuit in bacteria, the production and releasing of lactate increase evidently, and ldhDARSdR-lldP is the most efficient one. In summary, our engineered bacteria, Synechocystis sp. PCC 6803(cyanobacteria), do achieve our goal to provide lactate to Shewanella.

*![](https://static.igem.org/mediawiki/2018/6/63/T--HUST-China--2018-zhaozehong01.png)*

### **1.2 Rhodopseudomonas palustris (Rps)**

### **1.2.1 Verification of gene expression**

We transformed pMG105-PpckA-RBS-mleS-TT, pMG105-PpckA-RBS-lldP-TT, pMG105-PpckA-RBS-ldhA-TT, pMG105-PpckA-RBS-mleS-lldP-TT, pMG105-PpckA-RBS-lldP-RBS-ldhA-TT, pMG105-PpckA-RBS-ldhA-RBS-lldP-TT, pMG105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-TT into the E.coli BL21. And then, we do Real-Time Quantitative PCR to verificate targeted gene (Figure 4).

(A)

![](https://static.igem.org/mediawiki/2018/archive/2/20/20181017212558%21T--HUST-China--2018-result-pic1.png)

(B)

![](https://static.igem.org/mediawiki/2018/archive/2/20/20181017212625%21T--HUST-China--2018-result-pic1.png)

(C)

![](https://static.igem.org/mediawiki/2018/2/20/T--HUST-China--2018-result-pic1.png)

Figure 4. Relative expression level of targeted genes in E.coli BL21. We choose mleS(malate dehydrogenase, the conversion of malic acid to L-lactate), lldP(L-lactate permease, the lactate is transported out of the cell) and ldhA(fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate) as the reference genes and pMG105 as the standard quantity. (A). The expression level of mleS. (B). The expression level of lldP. (C). The expression level of ldhA. We could find that mleS, lldP and ldhA have a higher expression compared with pMG105. It means every genes can express efficiently.

Figure 4 shows that our genes express successfully on mRNA level.

### **1.2.2 Detection of Lactate**

We detect lactate production of every gene circuits in E.coli BL21 after incubating for 4h (Figure 5).

![](https://static.igem.org/mediawiki/2018/a/af/T--HUST-China--2018-result-rusuan04.png)

Figure 5. LB is a kind of medium to incubate bacterial. We detect lactate content of LB and the others. pMG105 has the lowest lactate content because pMG105 doesn’t have any gene to help produce lactate. It proves that all of our gene circuits have ability to produce lactate. Besides, Compared with ldhA and lldP, ldhA-lldP has a better production efficiency. Compared with ldhA-lldP, lldP-ldhA has a better production efficiency. Compared with ldhA-lldP, mleS-lldP has a better production efficiency. Compared with other gene circuits, mleS-lldP-ldhA has a better production efficiency. The result proves our modeling is correct.
 The figure 5 shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieve the goal to help strains produce lactate.

### **Part2: Electrogenic microorganism system**

*![](https://static.igem.org/mediawiki/2018/0/0c/T--HUST-China--2018-xiwa01.png)*

### **Shewanella Oneidensis**

### **2.1 Lactate utilization**

Since lactate is the carbon source of Shewanella Oneidensis MR-1, overexpression of lactate dehydrogenase might help the bacteria to utilize lactate more efficiently and produce more electricity. We chose dld(encodes D-lactate dehydrogenase) and lldEFG(encodes L-lactate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.

### 2.1.1 Gene Expression

To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.

![](https://static.igem.org/mediawiki/2018/a/a1/T--HUST-China--2018-result-fig1.png)

Figure 6.(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.

As we can see from figure 6, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.

![](https://static.igem.org/mediawiki/2018/5/5d/T--HUST-China--2018-result-fig2.png)

Figure 7. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.

It could be demonstrated that targeted genes could be expressed in the engineered cells. More lactate has been utilized by engineered bacteria, which helps to produce more electricty.

### **2.2.2 NADH production**

NADH is an important part for electrogenesis as it is one of the most important electron carriers in the cell. We hypothesized that more NADH produced by the cell would cause more electricity to be generated. Thus, we chose gapA(encodes glyceraldehyde-3-phosphate dehydrogenase), mdh(encodes NAD-dependent malate dehydrogenase), pflB(encodes pyruvate formate-lyase) and fdh(encodes formate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.

(1). Gene Expression

To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.

(A)

![](https://static.igem.org/mediawiki/2018/b/bf/T--HUST-China--2018-result-fig3.png)

(B)

![](https://static.igem.org/mediawiki/2018/8/86/T--HUST-China--2018-result-fig4.png)

(C)

![](https://static.igem.org/mediawiki/2018/5/51/T--HUST-China--2018-result-fig5.png)

(D)

![](https://static.igem.org/mediawiki/2018/a/a2/T--HUST-China--2018-result-fig6.png)

(E)

![](https://static.igem.org/mediawiki/2018/f/f9/T--HUST-China--2018-result-fig7.png)

Figure 8. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1. We chose gyrB(encodes DNA gyrase B) as the reference genes and 1 as the standard quantity. (A). The expression level of pYYDT-gapA, there was no signal in bacteria which contained pYYDT. (B). The expression level of pYYDT-mdh. (C). The expression level of pYYDT-gapA-mdh. (D). The expression level of pYYDT-pflB. (E). The expression level of pYYDT-pflB-fdh.

As figure 8 shows, gapA, mdh, pflB and fdh could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.

(2). Electrogenesis

By comparing the ability of producing electricity, we might find out whether pYYDT-gapA-mdh and pYYDT-pflB-fdh could effectively help Shewanella to produce more electricity.

![](https://static.igem.org/mediawiki/2018/0/0d/T--HUST-China--2018-result-fig8.png)

Figure 9. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.

It could be demonstrated that targeted genes could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.

### **Part3: Whole design**

*![](https://static.igem.org/mediawiki/2018/9/98/T--HUST-China--2018-coin17.png)*

### 3.1 Pre-experiment of electrogenesis

In the formal experiment. We compared the ability of electricity production between blank medium, Shewanella Oneidensis MR-1 and E.coli to make sure that Shewanella could produce electricity in our device.

![](https://static.igem.org/mediawiki/2018/4/44/T--HUST-China--2018-pre_experienment.png)

Figure 10. The comparison of ability of electricity production between wild type Shewanella Oneidensis MR-1 and E.coli.

We can find that Shewanella could produce electricity and the amount of electricity produced by Shewanella is much more than E.coli.

### 3.2 Lactate contrast

Then we designed two groups in order to explore the ability of electricity production when we added lactate in Shewanella Oneidensis MR-1.

![](https://static.igem.org/mediawiki/2018/d/d7/T--HUST-China--2018-result_kk_figure_4_1.png)

Figure 11. The influence when adding lactate in wild type Shewanella Oneidensis MR-1.

According to our experiments, we can make a conclusion: compared with medium that doesn’t add lactate, Shewanella oneidensis generates electricity more efficiently in lactate-added medium.

### 3.3 The difference between carbon rods and carbon cloth

We used carbon rods as electrodes at first. However, we were told by our adviser that the carbon cloth might have better effect for electrogenesis since more bacteria could attach on it.

![](https://static.igem.org/mediawiki/2018/2/25/T--HUST-China--2018-result-xiwachandian03.png)

Figure 12. The comparison of electricity production between using carbon rod and carbon cloth as the electrode.

It is easy to draw the conclusion that the carbon cloth has better effect than carbon rods as electrode.

### 3.4 The influence of oxygen on electrogenesis

During searching, we found that Shewanella Oneidensis MR-1 had commonly been used in electrogenesis at anaerobic condition. Since we chose Synechocystis PCC6803 as the lactate producer, the oxygen produced by Synechocystis PCC6803 could not be avoided. So we did this group of electrogenesis experiment to find out whether the oxygen would have a negative influence on producing electricity.

![](https://static.igem.org/mediawiki/2018/f/f5/T--HUST-China--2018-%E6%9C%89%E6%B0%A7%E6%97%A0%E6%B0%A7.png)

Figure 13. The comparison of electricity production between anaerobic circumstance and aerobic circumstance.

Shewanella could produce more electricity at anaerobic circumstance, which means that Synechocystis PCC6803 is not an ideal lactate producer in this electrogenesis system.

### 3.5 Contrast the symbiotic effect of wild-type strains`

We tried to compare the effects of Synechocystis PCC6803 and Rhodopseudomonas palustris by constructing a MFC system.

![](https://static.igem.org/mediawiki/2018/c/cc/T--HUST-China--2018-result-xiwachandian01.png)

Figure 14. The comparison of electricity production between Synechocystis PCC6803 and Rhodopseudomonas palustris

The figure shows that the effect of Rhodopseudomonas palustris is better than Synechocystis PCC6803. We believe that the oxygen produced by Synechocystis PCC6803 mainly reduces the electricity production effect.

### 3.6 Functional verification of engineered Synechocystis PCC6803

![](https://static.igem.org/mediawiki/2018/2/22/T--HUST-China--2018-result-xiwachandian02.png)

Figure 15. The comparison of electricity production between wild type and engineered Synechocystis PCC6803

In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.

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