iGEM 2023 — Team hust-china

![](https://static.igem.wiki/teams/4595/wiki/home/logo.png)

* [Home](index.html)
* Project

  [Description](description)
  [Safety](safety)
  [Proposed
  Implementation](implementation)
* Wet Lab

  [Design](design)
  [Engineering](engineering)
  [Protocol](protocol)
  [Results](results)
  [Notebook](notebook)
  [Parts](parts)
* [Hardware](hardware)
* HP

  [Human
  Practices](human-practices)
  [Education&
  Communication](education)
* Team

  [Contribution](contribution)
  [Members](members)
  [Attributions](attributions)

[![](https://2022.igem.wiki/hust-china/static/arrow-bar-down.svg)](#sectitle1)

#### content

* [Molecular Experiment](#sectitle1)
* [Expression Experiment](#sectitle2)

# Molecular Experiment

## 6.30-7.1

Transform plasmids PUC57-slr0168(vector) and PUC57-slr2030(vector) to *E. coli* TOP10.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate *E. coli* PUC57-slr0168(vector), PUC57-slr2030(vector).

![Figure 1. Left: The PCR identification result of PUC57-slr0168(vector),Right: The PCR identificationotebn result of PUC57-slr2030(vector)](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure1.png)
*Fig 1. Left: The PCR identification result of PUC57-slr0168(vector)

Right: The PCR identificationotebn result of PUC57-slr2030(vector)*

Purification of fragments PUC57-slr0168(vector) and PUC57-slr2030(vector)

Preparation of *E. coli* WM3064 competent cell.

## 7.3-7.4

Transform plasmids ldhA-lldP-omcs to *E. coli* TOP10.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate *E. coli* ldhA-lldP-omcs.

![Figure 2 Left: The PCR identification result of ldhA-lldP-omcs,Right: The PCR identification result of ldhA-lldP-omcs](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure2.png)
*Fig 2. Left: The PCR identification result of ldhA-lldP-omcs

Right: The PCR identification result of ldhA-lldP-omcs*

Purification of the fragments ldhA-lldP-omcs and omcs.notebook

## 7.6-7.7

Transform plasmids (PYYDT(vector), ycel-pncB and nadE-nadD-nadM) to *E. coli* TOP10.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate *E. coli* PYYDT(vector), ycel-pncB and nadE-nadD-nadM.

## 7.8

Extract plasmids (PYYDT(vector), ycel-pncB and nadE-nadD-nadM).

PCR and electrophorese to confirm.

![Figure 3 Left: The PCR identification result of PYYDT (vector),Right: The PCR identification result of ycel-pncB and nadE-nadD-nadM](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure3.png)
*Fig 3. Left: The PCR identification result of PYYDT (vector)

Right: The PCR identification result of ycel-pncB and nadE-nadD-nadM*

Purification of the fragments PYYDT(vector), ycel-pncB and nadE-nadD-nadM.

## 7.9

SOE PCR of ycel-pncB and nadE-nadD-nadM, and electrophorese to confirm.

![Figure 4 The PCR identification result of ycel-pncB and nadE-nadD-nadM](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure4.png)
*Fig 4. The PCR identification result of ycel-pncB and nadE-nadD-nadM*

Purification of the fragments ycel-pncB and nadE-nadD-nadM.

## 7.10-7.11

Transform plasmids (dsup-gshA-gshB) to *E. coli* TOP10.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate *E. coli* dsup-gshA-gshB.

## 7.12

Extract plasmids (dsup-gshA-gshB).

PCR and electrophorese to confirm.

![Figure 5 The PCR identification result of dsup-gshA-gshB](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure5.png)
*Fig 5. The PCR identification result of dsup-gshA-gshB*

Purification of dsup-gshA-gshB.

## 7.13-7.14

Transform plasmids (dsup-sodA-oprf) to *E. coli* TOP10.

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

Inoculate *E. coli* dsup-sodA-oprf.

## 7.15

Extract plasmids (dsup-sodA-oprf).

PCR and electrophorese to confirm.

![Figure 6 The PCR identification result of dsup-sodA-oprf](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure6.png)
*Fig 6. The PCR identification result of dsup-sodA-oprf*

Purification of dsup-sodA-oprf.

We have obtained all the plasmids and fragments we need and will next attempt to construct plasmids using homologous recombination.

## 7.16

Homologous recombination of ldhA-lldP-omcs and PUC57-slr0168(vector).

Homologous recombination of omcs and PUC57-slr0168(vector).

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 7.17

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

We didn’t succeed in connecting the fragments.

## 7.18

Homologous recombination of ldhA-lldP-omcs and PUC57-slr2030(vector).

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 7.19

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

We didn’t succeed in connecting the two fragments. It was suspected that there was a problem with the design of the primer homology arm and the primers were redesigned.

## 7.20-7.21

Inoculate E. coli ldhA-lldP-omcs and PUC57-slr0168(vector).

Extract plasmids ldhA-lldP-omcs and PUC57-slr0168(vector).

PCR with redesigned primers and electrophorese to confirm.

![The PCR identification result of ldhA-lldP-omcs, omcs and PUC57-slr0168(vector)](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure7.png)
*Fig 7. Left: The PCR identification result of ldhA-lldP-omcs

Middle: The PCR identification result of omcs

Right: The PCR identification result of PUC57-slr0168(vector)*

Purification of fragments ldhA-lldP-omcs, omcs and PUC57-slr0168(vector).

## 7.22-7.23

Inoculate E. coli dsup-gshA-gshB and PUC57-slr2030(vector).

Extract plasmids dsup-gshA-gshB and PUC57-slr2030(vector).

PCR with redesigned primers and electrophorese to confirm.

![The PCR identification result of dsup-gshA-gshB and PUC57-slr2030(vector)](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure8.png)
*Fig 8. Left: The PCR identification result of dsup-gshA-gshB

Right: The PCR identification result of PUC57-slr2030(vector)*

Purification of fragments dsup-gshA-gshB and PUC57-slr2030(vector).

## 7.24-7.25

Inoculate *E. coli* dsup-sodA-oprf, ycel-pncB, nadE-nadD-nadM and PYYDT(vector)

Extract plasmids dsup-sodA-oprf, ycel-pncB, nadE-nadD-nadM and PYYDT(vector).

PCR with redesigned primers and electrophorese to confirm.

![The PCR identification result of nadE-nadD-nadM, ycel-pncB, PYYDT(vector),and dsup-sodA-oprf](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure9.png)
*Fig 9. Left: The PCR identification result of nadE-nadD-nadM and ycel-pncBycel-pncB

Middle: The PCR identification result of PYYDT(vector)

Right: The PCR identification result of dsup-sodA-oprf*

Purification of fragments nadE-nadD-nadM, ycel-pncB, PYYDT(vector),and dsup-sodA-oprf.

## 7.26

SOE PCR of ycel-pncB and nadE-nadD-nadM, and electrophorese to confirm.

![The PCR identification result of ycel-pncB and nadE-nadD-nadM](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure10.png)
*Fig 10. The PCR identification result of ycel-pncB and nadE-nadD-nadM*

Purification of fragments ycel-pncB and nadE-nadD-nadM.

We have obtained all the plasmids and fragments we need by PCR with redesigned primers and will next attempt to construct plasmids using homologous recombination.

## 7.27

Homologous recombination of ldhA-lldP-omcs and PUC57-slr0168(vector).

Homologous recombination of omcs and PUC57-slr0168(vector).

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 7.28

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid omcs (Ppsba2-omcs-Tpsbc)](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure11.png)
*Fig 11. The PCR identification result of plasmid omcs (Ppsba2-omcs-Tpsbc)*

Plasmid omcs (Ppsba2-omcs-Tpsbc) was constructed successfully. Combined with water and glycerol, preserve.

We didn't succeed in connecting the ldhA-lldP-omcs and PUC57-slr0168(vector).

## 7.29

Homologous recombination of dsup-gshA-gshB and PUC57-slr2030(vector).

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 7.30

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

We didn’t succeed in connecting the dsup-gshA-gshB and PUC57-slr2030(vector).

## 7.31

Homologous recombination of ycel-pncB-nadE-nadD-nadM and PYYDT(vector).

Homologous recombination of ycel-pncB and PYYDT(vector).

Homologous recombination of nadE-nadD-nadM and PYYDT(vector).

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 8.1

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid ycel-pncB（Ptac-ycel-pncB-TrrnB T1） and nadE-nadD-nadM （Ptac-nadE-nadD-nadM-TrrnB T1）](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure12.png)
*Fig 12. Left: The PCR identification result of plasmid ycel-pncB（Ptac-ycel-pncB-TrrnB T1）and nadE-nadD-nadM （Ptac-nadE-nadD-nadM-TrrnB T1）*

Plasmid ycel-pncB（Ptac-ycel-pncB-TrrnB T1）and nadE-nadD-nadM （Ptac-nadE-nadD-nadM-TrrnB T1）were constructed successfully. Combined with water and glycerol, preserve.

## 8.2

Homologous recombination of dsup-sodA-oprf and PYYDT(vector.

Transform the plasmids (homologous recombination) to *E. coli* TOP10.

## 8.3

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

We didn’t succeed in connecting the dsup-sodA-oprf and PYYDT(vector).

Through homologous recombination, we constructed the plasmids omcs, ycel-pncB, nadE-nadD-nadM. For the remaining plasmids that were not constructed, we will try to construct them by double enzymes digestion and enzyme ligation.

## 8.4

Double enzymes digestion of ldhA-lldP-omcs and PUC57-slr0168(vector).

Ligating ldhA-lldP-omcs with PUC57-slr0168(vector).

Transform the plasmids (ligation) to *E. coli* TOP10.

## 8.5

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid pLactate (Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc）](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure13.png)
*Fig 13. The PCR identification result of plasmid pLactate (Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc）*

Plasmid pLactate (Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc）was constructed successfully. Combined with water and glycerol, preserve.

## 8.6

Double enzymes digestion of dsup-gshA-gshB and PUC57-slr2030(vector).

Ligating dsup-gshA-gshB with PUC57-slr2030(vector).

Transform the plasmids (ligation) to *E. coli* TOP10.

## 8.7

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid pResistance6803 (Pcpc560-dsup-gshA-gshB-Tpsbc-Ppsba2-slr2030-Tpsbc）](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure14.png)
*Fig 14. The PCR identification result of plasmid pResistance6803 (Pcpc560-dsup-gshA-gshB-Tpsbc-Ppsba2-slr2030-Tpsbc）*

Plasmid pResistance6803 (Pcpc560-dsup-gshA-gshB-Tpsbc-Ppsba2-slr2030-Tpsbc）was constructed successfully. Combined with water and glycerol, preserve.

## 8.8

Ligating ycel-pncB-nadE-nadD-nadM with PYYDT(vector).

Transform the plasmids (ligation) to *E. coli* TOP10.

## 8.9

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid pElectricty（Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1）](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure15.png)
*Fig 15. The PCR identification result of plasmid pElectricty（Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1）*

Plasmid pElectricty（Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1）was constructed successfully. Combined with water and glycerol, preserve.

## 8.10

Double enzymes digestion of dsup-sodA-oprf.

Ligating dsup-sodA-oprf with PYYDT(vector).

Transform the plasmids (ligation) to *E. coli* TOP10.

## 8.11

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.

![The PCR identification result of plasmid  pResistance MR-1（Ptac-dsup-sodA-oprf-TrrnB T1）](https://static.igem.wiki/teams/4595/wiki/notebook/notebook-figure16.png)
*Fig 16. The PCR identification result of plasmid pResistance MR-1（Ptac-dsup-sodA-oprf-TrrnB T1）*

Plasmid pResistance MR-1（Ptac-dsup-sodA-oprf-TrrnB T1）was constructed successfully. Combined with water and glycerol, preserve.

The plasmids pLactate, omcs, pElectricty, nadE-nadD-nadM, ycel-pncB and pResistance MR-1 have been constructed and transformed into E.coil TOP10. Next the plasmids pElectricty, nadE-nadD-nadM, ycel-pncB with pResistance MR-1 will be transformed into *E.coil* WM3064.

## 8.12-8.13

Inoculate E. coli pElectricty (TOP10), nadE-nadD-nadM (TOP10), ycel-pncB (TOP10).

Extract plasmids（pElectricty、nadE-nadD-nadM、ycel-pncB）.

Transform plasmids (pElectricty、nadE-nadD-nadM、ycel-pncB) to *E. coli* WM3064.

## 8.14

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

## 8.15-8.16

Inoculate *E.coli* pResistance MR-1 (TOP10).

Extract plasmids (pResistance MR-1).

Transform plasmids (pResistance MR-1) to *E. coli* WM3064.

## 8.17

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

## 8.18-8.19

Inoculate *E.coli* pResistance MR-1 (TOP10).

Extract plasmids (pResistance MR-1).

Transform plasmids (pResistance MR-1) to *E. coli* WM3064.

## 8.20

Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.

# Expression Experiment

## 8.20-8.23

Transfer the plasmids pElectricty、ycel-pncB、nadE-nadD-nadM into *Shewanella oneidensis* MR-1 cells separately by conjugating with *E.coli* WM3064.

## 8.24-8.25

Pick the bacterial plaque and expand the *Shewanella oneidensis* MR-1 after conjugal transfer.

## 8.26

Cell disruption of wild-type and target gene (pElectricty、ycel-pncB、nadE-nadD-nadM) introduced *Shewanella oneidensis* MR-1.

## 8.27

Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution, and the possibility of electricity generation was preliminarily verified. It was found that the protein was successfully expressed except nadE-nadD-nadM.

## 8.27-8.28

Transfer the plasmids (nadE-nadD-nadM) into *Shewanella oneidensis* MR-1 cells by conjugating with E.coil WM3064.

## 8.29

Pick the bacterial plaque and expand the *Shewanella oneidensis* MR-1 after conjugal transfer.

## 8.30

Cell disruption of wild-type and target gene (nadE-nadD-nadM) introduced *Shewanella oneidensis* MR-1.

Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution, and the possibility of electricity generation was preliminarily verified. It was found that nadE nadD nadM was successfully transformed.

## 8.30-9.10

Transfer genes pLactate, omcs, and pResistance6803 into *Synechocystis*  sp. PCC 6803and perform transformation validation. Expand the successfully transformed *Synechocystis*  sp. PCC 6803.

## 9.10

Cell disruption of wild-type and target gene introduced *Synechocystis*  sp. PCC 6803.

## 9.11

Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution.

## 9.12-9.13

Measure the lactate production of *Synechocystis*  sp. PCC 6803 with the target gene, then organize and analyze the data.

## 9.13-9.15

Determination of NADH and NADH/NAD+in *Synechocystis*  sp. PCC 6803 with different plasmids and target gene introduced *Shewanella oneidensis* MR-1.

## 9.16-9.18

Measure the electricity production ability of *Shewanella oneidensis* MR-1 at different rotational speeds, temperatures, and bacterial concentrations after conjugation transfer.

## 9.19-9.21

Add fluorescent dyes such as riboflavin to *Shewanella oneidensis* MR-1, and retest NADH and NADH/NAD+, as well as electricity production capacity.

## 9.21-9.28

Measure the OD600 of *Shewanella oneidensis* MR-1 and OD730 of *Synechocystis*  sp. PCC 6803 introduced with different target genes, and plot their growth curves.

## 9.21-9.28

Configure Mbg11 culture medium to cultivate *Shewanella oneidensis* MR-1 and determine the growth curve of *Shewanella oneidensis* MR-1.

## 9.28-10.4

Explore the co cultivation conditions of *Synechocystis*  sp. PCC 6803 and *Shewanella oneidensis* MR-1 (first analyze whether there is growth inhibition or toxicity to each other, and then explore the electricity production ability of the two under different proportions of inoculation), and draw their growth curves.

![arrow-down-square](https://2022.igem.wiki/hust-china/static/arrow-down-square.svg)
![arrow-up-square](https://2022.igem.wiki/hust-china/static/arrow-up-square.svg)

Access Wiki Tools
View Source Code
![]()

![](https://static.igem.wiki/teams/4595/wiki/home/logo.png)

* [Home](index.html)
* Project

  [Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
  [Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)
  [Proposed
  Implementation](implementation)
* Wet Lab

  [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
  [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
  [Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
  [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)
  [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
  [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)
* [Hardware](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D)
* HP

  [Human
  Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
  [Education&
  Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)
* Team

  [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
  [Members](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)
  [Attributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

[![](https://2022.igem.wiki/hust-china/static/arrow-bar-down.svg)](#sectitle1)

#### content

* [Introduction](#sectitle1)
* [CYCLE 1: constructed by homologous recombination method](#sectitle2)
* [CYCLE 2: constructed by double enzyme digestion and enzyme ligation method](#sectitle3)
* [CYCLE 3: Expression validation](#sectitle4)

# Introduction

We hope to build a co-culture system of  *Synechocystis*  sp. PCC 6803 and *S. oneidensis* MR-1, which can use solar energy to fix carbon dioxide to generate electricity. Therefore, we designed several components and four system paths to achieve this. In the build phase, we first used homologous recombination to build these pathways but we encountered some problems. Through continuous trials and summaries, we switched to using the double enzyme digestion and enzyme ligation to construct our pathway, and finally achieved certain results. Our experiments would provide some experience for subsequent teams.

# CYCLE 1: constructed by homologous recombination method

## 1.1 Design

To construct a well-functioning system pathway, we hope to utilize four plasmids to improve the efficiency of the electricity production of *S. oneidensis* MR-1, enhance the intensity of  *Synechocystis*  sp. PCC 6803 photosynthesis, and increase the antioxidant activity of  *Synechocystis*  sp. PCC 6803 and the stress resistance of *S. oneidensis* MR-1 respectively. Based on existing parts with independent functions, we achieve our goal by connecting multiple components to complete plasmids to form a fully functional system pathway.

We wanted to build our plasmid by using homologous recombination. Therefore, we introduce homologous arms into the components when building individual elements, which is the basis of the entire pathways construction.

First, In the pLactate pathway, we begin by incorporating the first homologous arm downstream of the slr0168 vector fragment, which includes the ampicillin-resistant gene, and upstream of ldhA-lldP-omcs. Following that, a second homologous arm is introduced downstream of ldhA-lldP-omcs and upstream of the slr0168 vector fragment.

In the pResistance6803 pathway, we add the first homologous arm to the downstream of the slr2030 vector fragment containing the ampicin and kana resistant genes and upstream of dsup-gshA-gshB. A second homology arm is then added downstream of dsup-gshA-gshB and upstream of the slr2030 vector fragment.

In the pResistance MR-1 pathway, we add the first homologous arm to the downstream of the PYYDT vector fragment containing the kana resistance gene and upstream of dsup-sodA-oprf. A second homology arm is then added downstream of dsup-sodA-oprf and upstream of the PYYDT vector fragment.

In the pElectricty pathway, we add the first homologous arm to the downstream of the PYYDT vector fragment containing the kanamycin resistance gene and upstream of ycel-pncB. A second homology arm was added downstream of ycel-pncB and upstream of nadD-nadE-nadM, and a third homology arm was added downstream of nadD-nadE-nadM and upstream of the PYYDT vector fragment. Homologous arms are added to different gene fragments by PCR using different primers. In principle, these specific homologous arms ensure our experimental feasibility.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/1.png)
*Fig 1. The diagram of constructing the plasmid inserted with pLactate by homologous recombination*
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/presistance6803.png)
*Fig 2. The diagram of constructing the plasmid inserted with pResistance6803 by homologous recombination*

## 1.2 Build

Based on the principle of seamless cloning, we tried to use homologous recombinase to attach multiple fragments directly into the vector to form a complete plasmid.

Take the pElectricty system pathway as an example: first, the linearized vector PYYDT and gene fragments - ycel-pncB and nadD-nadE-nadM were obtained by PCR. They were then validated and purified by gel extraction. Finally, homologous recombinase was used to directly connect the linearized vector PYYDT with two fragments of ycel-pncB and nadD-nadE-nadM to construct a complete system pathway. We then transfered the homologous recombinant product into *E. coli* TOP10 and seed into LB plates containing 50 μg/mL kanamycin for screening. When single colonies appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of bacterial solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

## 1.3 Result

We tried to construct our pathway through multi-fragment homologous recombination. After several attempts, we successfully constructed plasmids omcs, ycel-pncB and nadE-nadD-nadM.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/tycz.png)
*Fig 3. The left figure is the PCR identification result of omcs plasmid.It shows that there is a bright band at about 2000 bp, and the length of omcs plasmid is 1867 bp, which proves that we have successfully constructed the plasmid of omcs system pathway.*
*The right figure is the PCR identification result of ycel-pncB plasmid and nadE-nadD-nadM plasmid.It shows that there is a bright band at about 2500 bp and a bright band at about 3000 bp. The length of ycel-pncB plasmid is 2601 bp and the length of nadE-nadD-nadM plasmid is 2820 bp , which proves that we have successfully constructed the plasmid of ycel-pncB system pathway and the plasmid of nadE-nadD-nadM system pathway.*

## 1.4 Experience

After several attempts, we found that the direct use of multi-fragment homologous recombination is not suitable for most pathways, so we carefully analyzed and summarized the reasons for the failures. We believe that the success rate of homologous recombination is low due to the large number of genes, the huge number of bases and the complex topology contained in the pResistance MR-1, pLactate, pResistance 6803, pElectricity pathways. Therefore, we tried to convert to the method of enzyme digestion and ligation to solve this problem.

# CYCLE 2: constructed by double enzyme digestion and enzyme ligation method

## 2.1 Design

After many failed attempts, we decided to try double enzyme digestion and enzyme ligation to construct our plasmids.

We first perform a double enzyme digestion reaction to obtain multiple fragments and vectors with the same sticky ends, and then ligate the carriers and fragments containing the same sticky ends together through an enzyme ligation reaction. We hope it will solve the problem of low success rate of homologous recombination in dealing with multi-fragment and large-fragment at this stage.

In the first step, we use the double enzyme digestion method to obtain the target gene fragments and the vector fragment with sticky ends. We first culture the basal plasmid-containing strain and extract the plasmid, then add the digestion site to the target gene fragment and the vector fragment by PCR, and then obtain the target gene fragment and the vector fragment with the same sticky end by double enzyme digestion. For dsup-gshA-gshB fragments for  *Synechocystis*  sp. PCC 6803 and ycel-pncB-nadE- nadD-nadM fragments for *S. oneidensis* MR-1, we directly cleavage the extracted basal plasmid by double enzyme digestion to obtain gene fragments with sticky ends, For the ldhA-lldP-omcs fragment for  *Synechocystis*  sp. PCC 6803 and the dsup-sodA-oprf fragment for *S. oneidensis* MR-1, we first PCR the extracted plasmid to obtain the target gene fragments, and then obtain the fragment with sticky end by double enzyme digestion.

In the second step, we use enzyme ligation to link the target gene fragment and its corresponding vector fragment with the same sticky end together, and finally construct a complete plasmid.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/5.png)
*Fig 4. The diagram of constructing the plasmid inserted with pLactate by double enzyme digestion and enzyme ligation*
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/6.png)
*Fig 5. The diagram of constructing the plasmid inserted with pResistance6803 by double enzyme digestion and enzyme ligation*
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/7.png)
*Fig 6. The diagram of constructing the plasmid inserted with pResistance MR-1 by double enzyme digestion and enzyme ligation*
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/8.png)
*Fig 7. The diagram of constructing the plasmid inserted with pElectricity by double enzyme digestion and enzyme ligation*

## 2.2 Build

Our basic process of constructing a complete plasmid is: extract the plasmid, perform PCR to obtain the fragment containing the target gene, obtain the fragment of the target gene with sticky ends through double enzyme digestion, and use the enzyme ligation reaction to link the gene of interest to the vector.

Take the pLactate system pathway as an example: we first obtain linearized vector fragments containing ampicure resistance by PCR, and then obtain gene fragments and vector fragments by gel extraction, then verify and purify gene fragments and vector fragments. We then double-enzyme-digested the fragments with restriction enzymes SpeI and EcorI to obtain gene and vector fragments with identical sticky ends. Finally, we used an enzyme ligation reaction to ligate a fragment containing the gene ldhA-lldP-omcs with the vector fragment corresponding to it to construct a complete plasmid. We then transferred the constructed plasmid into *E. coli* TOP10 and seeded into LB plates containing 100 μg/mL ampicin for screening. When single colonies appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of bacterial solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

## 2.3 Result

After several attempts, we successfully constructed plasmids pLactate, pResistance6803, pElectricity, pResistance MR-1.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/mqml1.png)
*Fig 8. The left figure is the result of PCR identification of pLactate plasmid.It shows that there is a bright band at about 5000 bp, and the length of pLactate plasmid is 5479 bp, which proves that we have successfully constructed the plasmid of pLactate system pathway.*
*The right figure is the result of PCR identification of pResistance6803 plasmid.It shows that there is a bright band at about 5000 bp, and the length of pResistance6803 plasmid is 4746 bp, which proves that we have successfully constructed the plasmid of pResistance6803 system pathway.*
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/mqml2.png)
*Fig 9. The left figure is the result of PCR identification of pElectricity plasmid.It shows that there is a bright band at about 5000 bp, and the length of pElectricity plasmid is 5736 bp, which proves that we have successfully constructed the plasmid of pElectricity system pathway.*
*The right figure is the result of PCR identification of pResistance MR-1 plasmid.It shows that there is a bright band at about 3000 bp, and the length of pResistance MR-1 plasmid is 3291 bp, which proves that we have successfully constructed the plasmid of pResistanceMR-1 system pathway.*

## 2.4 Experience

At first, we encountered the problem that the digestion reaction could not be carried out due to the low concentration of the recovered PCR fragments. After several attempts, we found that the extraction of PCR fragment products, the timing and temperature of the double enzyme digestion reaction and the enzyme ligation reaction had a great influence on the success rate of plasmid construction. Following numerous setbacks, we meticulously analyzed and documented the underlying causes. After conducting a series of experiments, we successfully extracted the necessary fragments and vectors from fluid samples, maximizing their concentrations for double enzyme digestion. After double enzyme digestion, we obtained the double enzyme digestion product by gel extraction to ensure the purity of the fragment. At the same time, we also continuously adjusted the reaction temperature and narrowed The time range of the reaction, and finally found the most suitable reaction time and temperature system. After a series of difficulties, we figured out an optimally constructed system and obtained the corresponding complete plasmid.

Electrico sisters is of great significance to solve the energy crisis and protect the global environment, encouraging us to use synthetic biology to make some contributions in this regard. We will continue to carry out this topic and are willing to do our part for the development of synthetic biology.

# CYCLE 3: Expression validation

## 3.1 *Synechocystis* sp. PCC 6803

### 3.1.1 Design

We hope to figure out whether the growth cycle of  *Synechocystis*  sp. PCC 6803 engineered bacteria is shorter than that of wild-type  *Synechocystis*  sp. PCC 6803. Therefore, we detect the growth of  *Synechocystis*  sp. PCC 6803 and plot its growth curve. At the same time, we verify whether the efficiency of the strain we constructed achieves our expectations by detecting the concentration of expression products.

### 3.1.2 Build

First, we culture the *E. coli* TOP10 strain containing the correct plasmid and extracted the plasmid. Next, we transfer the extract plasmid into  *Synechocystis*  sp. PCC 6803. The experimental operation is: we use BG11 liquid medium to resuspend and wash wild-type  *Synechocystis*  sp. PCC 6803 twice, take an appropriate amount of  *Synechocystis*  sp. PCC 6803 solution and add an appropriate amount of plasmid, stand and activate it for a period of time, apply the bacterial solution to BG11 plate with corresponding resistant. When the transformant appears on the surface of the solid medium, we pick out the transformant for PCR verification. After observing the correct bands, we mix 800 μl of  *Synechocystis*  sp. PCC 6803 solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

### 3.1.3 Test

We verified that our plasmid was successfully transferred to  *Synechocystis*  sp. PCC 6803 by colony PCR. The specific method is to pick the selected single colony of  *Synechocystis*  sp. PCC 6803 and delineat for expanded culture on plates. After more colonies grew up, we pick them into liquid BG11 medium to get enough bacteria solution and extract the  *Synechocystis*  sp. PCC 6803 genome. And conduct PCR verification after the bacteria is amplified for avoiding PCR failure caused by the engineering bacterium concentration is too low, or PCR success but strain amplification failure cases.
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig10.png)
*Fig 10. The PCR of pLactate, omcs,and presistance6803 colonies all had bright and correct bands, which proved that the transformation was successful.*

After colony PCR identified bright and correct bands, we preserved them for expanded culture and induction of expression. We examined the growth curves of wild-type *Synechocystis*  sp. PCC 6803 and  *Synechocystis*  sp. PCC 6803(omcs) in BG11 medium. Compared to  *Synechocystis*  sp. PCC 6803,  *Synechocystis*  sp. PCC 6803(omcs) exhibited a significant improvement in cell concentration at all stages, and there was no significant decrease in growth rate. This suggests that omcs can enhance the photosynthetic efficiency of PS1 by increasing ATP synthesis, which promots better growth of cells. It proved that the genes we transferred into *Synechocystis*  sp. PCC 6803 were finally effectively expressed.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig11.png)
*Fig 11. The growth curve of *Synechocystis*  sp. PCC 6803 and *Synechocystis*  sp. PCC 6803(omcs) in BG11.*

At the same time, we detected the amount of lactate produced by wild-type *Synechocystis*  sp. PCC 6803 and *Synechocystis*  sp. PCC 6803 engineering bacteria pLactate. Comparing the data of wild-type and engineered bacteria pLactate, we found that the amount of expression products of *Synechocystis*  sp. PCC 6803 engineering bacteria pLactate were higher than those of wild-type *Synechocystis*  sp. PCC 6803, demonstrating that the strain we have built works as expected.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig20.png)
*Fig 12. The left figure is the HPLC spectrogram of lactate standard product in different concentration.*
*The right figure is the standard curve of lactate.*
![](https://static.igem.wiki/teams/4595/wiki/results/results/fig21.png)
*Fig 13.The figure is the HPLC spectrogram of *Synechocystis*  sp. PCC 6803, *Synechocystis*  sp. PCC 6803(omcs) and 5mM Lactate standard product. There is a peak at 3.932 min both in *Synechocystis*  sp. PCC 6803(omcs) and 5mM Lactate standard product which represent lactate, and *Synechocystis*  sp. PCC 6803 don't have this peak. It indicated that *Synechocystis*  sp. PCC 6803(omcs) could product lactate while the wild type don't have this ability.*

## 3.2 *Shewanella oneidensis* MR-1

### 3.2.1 Design

We hope to verify that the strain we built worked as efficiently as expected by measuring the amount of the expression product NADH/NAD+. At the same time, we compared whether the ability to generate electricity of *S. oneidensis* MR-1 transferred with the plasmid we constructed was stronger than that of wild-type *S. oneidensis* MR-1 by measuring the efficiency of electricity production from *S. oneidensis* MR-1.

### 3.2.2 Build

We culture the strain containing the correct plasmid and extract the plasmid to transfer into *E. coli* WM3064. Subsequently, we transferred the plasmid we constructed into *S. oneidensis* MR-1 by conjugation between *E. coli* WM3064 and *S. oneidensis* MR-1. The experimental operation is: we inoculated an equal and appropriate amount of *E. coli* WM3064 with *S. oneidensis* MR-1 into 1 ml liquid LB medium containing 0.019 g/mL DAP and 50 μg/mL kanamycin for one hour, and finally inoculated the mixed bacteria solution on LB plates containing 50 μg/mL / mL of kanamycin. When single colonies of *S. oneidensis* MR-1 appeared on the surface of solid media, we singled out the single colonies for PCR validation. After observing the correct bands, we mixed 800 μl of *S. oneidensis* MR-1 solution with 800 μl of 1:1 mixture of glycerol and double distilled water and keep the seeds at -80℃.

### 3.2.3 Test

We verified that our plasmid was successfully transferred to *S. oneidensis* MR-1 by colony PCR. The specific method is to culture the selected single colony of *S. oneidensis* MR-1 in a shake flask containing 1ml liquid LB with Kana, and conduct PCR verification after the bacteria is amplified for avoiding PCR failure caused by the engineering bacterium concentration is too low, or PCR success but strain amplification failure cases.
![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig14.png)
*Fig 14. The PCR of pElectricity, ycel-pncB, nadE-nadD-nadM, presistanceMR-1 colonies all had bright and correct bands, which proved that the transformation was successful.*

After colony PCR identified bright and correct bands, we preserved them for expanded culture and induction of expression. We measured the amount of NADH/NAD+ produced by wild-type *S. oneidensis* MR-1 and *S. oneidensis* MR-1 with plasmid ycel-pncB or nadE-nadD-nadM. All bacteria were cultured in LB medium with Kana. When the OD600 of medium reached 0.6-0.8, we added 0.5mM IPTG to intrudce the target gene expression. Culture overnignt after introduing, and extract NAD(H/+) by NAD(H/+) extraction kit. Compared to the wild type,*S. oneidensis* MR-1(ycel-pncB) showed a 22.23% increase in NADH/NAD+ ratio, while the total amount of NADH/NAD+ in*S. oneidensis* MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that *S. oneidensis* MR-1(ycel-pncB) may contribute to the accelerated transport of niacin and nicotinamide for intracellular NADH synthesis, while *S. oneidensis* MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer, proving that the working efficiency of the strain we constructed achieved our expectations.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig15.jpg)
*Fig 15. The NAD(H/+) concentration of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadE-nadD-nadM).*

At the same time, we tested the electricity production of wild-type *S. oneidensis* MR-1 and *S. oneidensis* MR-1 with plasmid ycel-pncB or nadE-nadD-nadM. Comparing the electricity production of the two, we found that the voltage of the battery constructed by *S. oneidensis* MR-1 engineering bacteria ycel-pncB and nadE-nadD-nadM was higher than that of the battery constructed by wild-type *S. oneidensis* MR-1.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig16.png)
*Fig 16. The out put voltage of *S. oneidensis* MR-1,*S. oneidensis* MR-1(ycel-pncB), *S. oneidensis* MR-1(nadE-nadD-nadM).*

![arrow-down-square](https://2022.igem.wiki/hust-china/static/arrow-down-square.svg)
![arrow-up-square](https://2022.igem.wiki/hust-china/static/arrow-up-square.svg)

Access Wiki Tools
View Source Code
![]()

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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).

## Inspirations

---

* [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)

{% endblock %}

![](https://static.igem.wiki/teams/4595/wiki/home/logo.png)

* [Home](index.html)
* Project

  [Description](description)
  [Safety](safety)
  [Proposed
  Implementation](implementation)
* Wet Lab

  [Design](design)
  [Engineering](engineering)
  [Protocol](protocol)
  [Results](results)
  [Notebook](notebook)
  [Parts](parts)
* [Hardware](hardware)
* HP

  [Human
  Practices](human-practices)
  [Education&
  Communication](education)
* Team

  [Contribution](contribution)
  [Members](members)
  [Attributions](attributions)

[![](https://2022.igem.wiki/hust-china/static/arrow-bar-down.svg)](#sectitle1)

#### content

* [Introduction](#sectitle1)
* [Plasmid amplification and reconstruction](#sectitle2)

+ [Plasmids for *Synechocystis*  sp. PCC 6803](#list1)
+ [Plasmids for *S. oneidensis* MR-1](#list2)

* [Strain construction](#sectitle3)
* [Expression Validation](#sectitle4)

+ [*Synechocystis*  sp. PCC 6803](#list3)
+ [*S. oneidensis* MR-1](#list4)
+ [Co-culture system](#list5)

# Section 1 : Introduction

Our project aimed to enable *Synechocystis*  sp. PCC 6803 to produce lactic acid at a high efficiency, enhance the electricity producing capacity of *S. oneidensis* MR-1, as well as improve the resistance of both bacteria. In order to achieve these goals, we construct mainly 4 plasmids: pLactate and pResistence6803 for *Synechocystis*  sp. PCC 6803, pElectricity and pResistance MR-1 for *S. oneidensis* MR-1. Besides 4 main plasmids, we also constructed 3 plasmids in order to test the function of genes more conveniently.

We transferred them into *Synechocystis*  sp. PCC 6803 and *S. oneidensis* MR-1 for expressing the proteins we needed. At the same time, in order to verify the efficiency of electricity production, we also carried out and verified our project from all aspects.

# Section 2 : Plasmid amplification and reconstruction

We first transformed the plasmids containing designed sequence synthesized by the company into Escherichia coli Top10 for amplification, so we have enough amount of plasmid to carry out subsequent experiments. Then colony PCR showed a successful transformation, and required plasmids were extracted for the next experiment.

We then obtained target fragments by means of PCR, and then constructed them into the plasmids we need.

## 2.1 Plasmids for *Synechocystis* sp. PCC 6803

We have 2 plasmids, pLactate and pResistance6803 for *Synechocystis*  sp. PCC 6803. We verified the size of each plasmids as well as all the fragments involved in constructing the plasmids. In addition to pLactate and pResistance6803, we also constructed plasmid Omcs to ensure the function of it individually.

pLactate includes LdhA enabling *Synechocystis*  sp. PCC 6803 to produce lactic acid by means of photosynthesis, Lldp to transform intracellular lactate to the extracellular as well as Omcs improving the efficiency of electricity transformation.

![](https://static.igem.wiki/teams/4595/wiki/results/results/gaitushu.png)
![](https://static.igem.wiki/teams/4595/wiki/results/results/1-gh.png)
*Fig 1. The PCR result of Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc.*

The bands of Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc(5000+bp) from PCR are identical to the theoretical lengths of 5479bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/2-g-1.png)
*Fig 2. The PCR result of Pcpc560-ldhA-lldP-Tpsbc.*

The bands of Pcpc560-ldhA-lldP-Tpsbc(3000+bp) from PCR are identical to the theoretical lengths of 3643bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/3-1-g-2-g-2.png)
![](https://static.igem.wiki/teams/4595/wiki/results/results/3-2-g-2-gh.png)
*Fig 3. The PCR result of Ppsba2-omcs-Tpsbc from pLactate(left) and Omcs(right).*

The bands of Ppsba2-omcs-Tpsbc(almost 2000bp) from PCR are identical to the theoretical lengths of 1866bp(from pLactate) and 1820bp(from Omcs) estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/0168wz.png)
*Fig 4. The PCR result of PUC57-slr0168(vector).*

The bands of PUC57-slr0168(vector)(almost 5000bp) from PCR are identical to the theoretical lengths of 4727bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pLactate and Omcs.

pResistance6803 is constructed to enhance the resistance of *Synechocystis*  sp. PCC 6803 under adversities, especially the antioxidant capacity and the ability to protect DNA from damages.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig2.png)
![](https://static.igem.wiki/teams/4595/wiki/results/results/5-lir.png)
*Fig 5. The PCR result of fragment PrbcL-dsup-Tpsbc-PrbcL-gshA-gshB-Tpsbc.*

The bands of PrbcL-dsup-Tpsbc-PrbcL-gshA-gshB-Tpsbc(almost 5000bp) from PCR are identical to the theoretical lengths of 4746 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/6-l-1.png)
*Fig 6. The PCR result of fragment PrbcL-dsup-Tpsbc.*

The bands of PrbcL-dsup-Tpsbc (almost 2000bp) from PCR are identical to the theoretical lengths of 1848 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/7-l-2.png)
*Fig 7. The PCR result of fragment PrbcL-gshA-gshB-Tpsbc.*

The bands of PrbcL-gshA-gshB-Tpsbc(almost 3000bp) from PCR are identical to the theoretical lengths of 2889 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/2030wz.png)
*Fig 8. The PCR result of fragment PUC57-slr2030.*

The bands of PUC57-slr2030(almost 5000bp) from PCR are identical to the theoretical lengths of 4633 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pResistance6803.

## 2.2 Plasmids for *S. oneidensis* MR-1

We have 2 plasmids, pElectrcity and pResistance MR-1 for *S. oneidensis* MR-1. We verified the size of each plasmids as well as all the fragments involved in constructing the plasmids . Besides pElectricity and pResistanceMR-1, we also constructed plasmids to ensure the function of ycel-pncB and nadE-nadD-nadM individually.

pElectricty is constructed with ycel-pncB and nadE-nadD-nadM, both can elevate the electricity production rate of *S. oneidensis* MR-1.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig3.png)
![](https://static.igem.wiki/teams/4595/wiki/results/results/9-cd.png)
*Fig 9. The PCR result of fragment Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1.*

The bands of Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1(5000+bp) from PCR are identical to the theoretical lengths of 5736 estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/gaituwz2.png)
*Fig 10. The PCR results of Ptac-ycel-pncB-TrrnB T1 and Ptac-nadE-nadD-nadM-TrrnB T1 from pElectricity.*

The bands of Ptac-ycel-pncB-TrrnB T1(2500+bp) and Ptac-nadE-nadD-nadM-TrrnB T1(about3000 bp) from PCR are identical to the theoretical lengths of 2624bp and 2820bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/11-c-1.png)
*Fig 11. The PCR results of fragment Ycel-PncB from ycel-pncB.*

The bands of Ycel-PncB(about 2500bp) from PCR are identical to the theoretical lengths of 2622bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/12-c-2.png)
*Fig 12. The PCR results of Ptac-nadE-nadD-nadM-TrrnB T1 from nadE-nadD-nadM.*

The bands of Ptac-nadE-nadD-nadM-TrrnB T1 (about 3000bp) from PCR are identical to the theoretical lengths of 2820bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/pyydtwz.png)
*Fig 13. The PCR results of PYYDT(vector).*

The band of PYYDT(vector) (5000+bp) from PCR is identical to the theoretical lengths of 5886bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had been successfully obtained.

All the results above showed us a success of the construction of pElecteicity, ycel-pncB and nadE-nadD-nadM.

pResistance MR-1 is constructed with dsup-sodA and oprf, which can lower the level of ROS in *S. oneidensis* MR-1 and avoid DNA damages, and the latter can assist in the entry and exit of riboflavin and improves the efficiency of electron transfer

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig4.png)
![](https://static.igem.wiki/teams/4595/wiki/results/results/14-sir.png)
*Fig 14. The PCR result of Ptac-dsup-sodA-oprf-TrrnB T1.*

The bands of Ptac-dsup-sodA-oprf-TrrnB T1(3000+bp) from PCR are identical to the theoretical lengths of 3291bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/15-s-12.png)
*Fig 15. The PCR result of fragment Ptac-dsup-sodA-TrrnB T1.*

The bands of Ptac-dsup-sodA-oprf-TrrnB T1(3000+bp) from PCR are identical to the theoretical lengths of 3291bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

![](https://static.igem.wiki/teams/4595/wiki/results/results/16-s-3.png)
*Fig 16. The PCR result of fragment Ptac-oprf-TrrnB T1.*

The bands of Ptac-oprf-TrrnB T1(1000+bp) from PCR are identical to the theoretical lengths of 1135bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these fragments had been successfully obtained.

The results above could demonstrate that these plasmids are correctly constructed, so we can transform pLactate, pResistence6803,and omcs into *Synechocystis*  sp. PCC 6803, while pElectricity, ycel-pncB, nadD-nadE-nadM and pResistance MR-1into *S. oneidensis* MR-1 for verification.

# Section 3 :Strain construction

We have successfully constructed all the target plasmids needed in the four loops, and this step really facilitates our subsequent experiments. At the same time, in order to facilitate the measurement of the function of some elements, we separately constructed plasmids (omcs, ycel-pncB, nadD-nadE-nadM) for some essential elements.

![](https://static.igem.wiki/teams/4595/wiki/results/results/results1.png)
*Fig 17. Colony PCR results of pLactate, Omcs and pResistence6803.*
![](https://static.igem.wiki/teams/4595/wiki/results/results/resultsjt2.png)
*Fig 18. Colony PCR results of target fragments in plasmids pElectricity, ycel-pncB, nadD-nadE-nadM and pResistenceMR-1.*

All the bands are identical to the theoretical sizes, demonstrating that these plasimds had been successfully transformed and verified.The results are as follows: PLactate: Pcpc560 - ldhA - lldP - Tpsbc - Ppsba2 - omcs -Tpsbc about 5400 bp , pResistance6803: PrbcL - dsup - Tpsbc - PrbcL - gshA - gshB -Tpsbc about 4100 bp , omcs: Ppsba2-omcs-Tpsbc about 1800bp, pElectricty:Ptac-ycel-pncB-TrrnB T1-Ptac-nadD-nadE-nadM-TrrNB T1 about 5700bp, pResistance MR-1: Ptac-dsup-soda-oprf-TrrnB T1 about 3200bp , ycel-pncB: Ptac-Ycel-PNCB-TrrnB T1 about 2500bp, nadD-nadE-nadM: Ptac-nadD-nadE-nadM-TrrNB T1 about 2800bp

The theoretical length of 5461bp, 4107bp,1809bp, 5666bp, 3229bp, 2564bp and 2820bp estimated by the design primers location (promoter to terminator) was the same, indicating that we successfully obtained the plasmid we needed.

The successful plasmids transformed into *Synechocystis*  sp. PCC 6803 and *S.oneidensis MR-1* was verified by colony PCR.

# Section 4 : Expression Validation

### 4.1 *Synechocystis* sp. PCC 6803

##### 4.1.1 Growth curve of *Synechocystis* sp. PCC 6803

We used BG11 to culture the engineered bacteria *Synechocystis*  sp. PCC 6803(omcs) and wild type respectively, and measured the OD730 every 24 hours. A growth curve is obtained as follows.

The results showed that compared to *Synechocystis*  sp. PCC 6803, *Synechocystis*  sp. PCC 6803(omcs) exhibited a significant improvement in cell concentration at all stages, and there was no significant decrease in growth rate. This suggests that omcs can enhance the photosynthetic efficiency of PSI by increasing ATP synthesis, which promots better growth of cells

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig11.png)
*Fig 19. The growth curve of *Synechocystis*  sp. PCC 6803 and *Synechocystis*  sp. PCC 6803(omcs) in BG11.*

##### 4.1.2 Lactate production verification

We want to co-culture *Synechocystis*  sp. PCC 6803 and *S. oneidensis* MR-1. The nutrient of *S.oneidensis MR-1* is lactate produced by *Synechocystis*  sp. PCC 6803. We tried to determine the production of lactate by HPLC to test the feasibility of the co-culture method.

We first measured the standard curve of lactate concentration and determined its retention time. The *Synechocystis* PCC6803(pLactate) cells were broken by freeze-thaw method, and the supernatant after centrifugation was taken for high performance liquid chromatography (HPLC).

A peak with almost the same retention time which is 3.932 min as the lactate standard substance was observed on the spectrum, which confirmed the successful expression of lactate in engineered bacteria. The peak area was compared with the standard curve, which can be seen that the lactate yield of our engineering *Synechocystis*  sp. PCC 6803 was 7.26mM, indicating that the gene we introduced could produce lactic acid stably and provide nutrition for *S. oneidensis* MR-1.

This result indicated the successful expression of the imported LdhA and LldP genes, and verified the feasibility of the co-culture system.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig20.png)
*Fig 20. The left figure is the HPLC spectrogram of lactate standard product in different concentration.*
*The right figure is the standard curve of lactate(0-10 mM).*
![](https://static.igem.wiki/teams/4595/wiki/results/results/fig21.png)
*Fig 21.The figure is the HPLC spectrogram of *Synechocystis*  sp. PCC 6803, *Synechocystis*  sp. PCC 6803(omcs) and 5mM Lactate standard product. There is a peak at 3.932 min both in *Synechocystis*  sp. PCC 6803(omcs) and 5mM Lactate standard product which represent lactate, and *Synechocystis*  sp. PCC 6803 don't have this peak. It indicated that *Synechocystis*  sp. PCC 6803(omcs) could product lactate while the wild type don't have this ability.*

### 4.2 *S. oneidensis* MR-1

##### 4.2.1 Determination of NADH/NAD+ concentration

To verify normal gene expression in *S. oneidensis* MR-1, we measured the NADH/NAD+ content of wild type and engineered strains ycel-pncB and nadD-nadE-nadM, respectively.

Compared to the wild type, *S. oneidensis* MR-1(ycel-pncB) showed a 22.23% increase in NAD(H/+) amount, while the total amount of NAD(H/+) in *S. oneidensis* MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, *S.oneidensis MR-1* (ycel-pncB) may contribute to the accelerated transport of niacin and nicotinamide for intracellular NADH synthesis, while *S. oneidensis* MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.

![](https://static.igem.wiki/teams/4595/wiki/engineering/engineering/fig15.jpg)
*Fig 22. The NAD(H/+) concentration of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadE-nadD-nadM).*

##### 4.2.2 Verification of electricity production of *S. oneidensis* MR-1

We built a microbial fuel cell device to test the electricity-producing performance of*S. oneidensis* MR-1.

We inoculated *S. oneidensis* MR-1 cultured overnight in 100 mL LB medium at 30℃ and 200 rpm to OD600 to 0.6-0.8. The cultured cells were collected by centrifuge and re-suspended in 140 mL anoditic solution. 50 μg/mL kana antibiotics and 18mM lactate were added to maintain constant culture conditions and 0.1 mM IPTG inducer to initiate gene expression. The two electrode chambers of MFC are separated by proton exchange membrane, using carbon cloth as cathode and anode electrodes, and then adding 140mL cathode solution to the cathode chamber, using 2kΩ external resistance to close the external circuit.

The specific battery structure of a complete MFC is as follows

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig22.jpg)
*Fig 23. The microbial fuel cell device we built.*

1) Testing the MFC

After construction the MFC, we use a digital multimeter to measure the output voltage of microbial fuel cell.

We measured the voltage of *S. oneidensis* MR-1(ycel-pncB), *S. oneidensis* MR-1(nadD-nadE-nadM) and compared them with the voltage of *S. oneidensis* MR-1. Measure a set of voltages every 6 hours and compare as follows.

The results showed that *S. oneidensis* MR-1(ycel-pncB) exhibited a significantly higher power output than the wild type before 30 hours, but experienced a noticeable decrease after 30 hours. It is speculated that this could be attributed to the ability of*S. oneidensis* MR-1(ycel-pncB) to accelerate the transport of niacin and nicotinamide for intracellular NADH synthesis, resulting in a higher power output before 30 hours.

However, due to the absence of exogenous niacin and nicotinamide supplementation, the availability of NADH synthesis precursors decreases, leading to a decline in intracellular NADH concentration and a slower electron transfer rate, ultimately resulting in reduced power generation efficiency.

In contrast, nadD-nadE-nadM exhibited significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output .

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig24.png)
*Fig 24. The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) when the anoditic solution is M9 buffer and 18mM lactate.*

2) Exogenous electron carrier

In our search for relevant papers, we learned that exogenous electron carrier such as photosensitivities and riboflavin contribute to extracellular electron transfer, thereby improving the efficiency of electricity generation. Therefore, we investigated the effect of addition of photosensitizers (5 μM Eosin Y, 5 μM Fluorescein) and 40 μM riboflavin on electricity production in *S. oneidensis* MR-1.

We use *S. oneidensis* MR-1(nadD-nadE-nadM) to try getting the highest voltage output by adding exogenous electron carrier including riboflavin and different photosensitizers. We constructed the MFC device and observed that riboflavin significantly increased the peak power output by 5.56 times after 20 hours with the highest voltage at 668mV, while the other exogenous electron carrier showed no significant change. This could be attributed to the optimization of electron carriers and enhanced electron transfer efficiency after the addition of riboflavin.

The results show that riboflavin is helpful in generating electricity.And considering the low cost of riboflavin, it can be applied in subsequent industrial production.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig25.png)
*Fig 25. The out put voltage of S.oneidensis MR-1(nadD-nadE-nadM)with different exogenous electron carrier.*

3) Practical application

In order to verify the practical application effect of the microbattery, we connected the microbattery containing the gene pathways of *S. oneidensis* MR-1, *S. oneidensis* MR-1(ycel-pncB), *S. oneidensis* MR-1(nadD-nadE-nadM)with a red LED bulb , respectively.

The results showed that the brightness of LED bulb did not change significantly after being connected with the *S. oneidensis* MR-1 micro battery, indicating that the *S. oneidensis* MR-1 micro battery did not reach the operating voltage of LED bulb, while the red LED bulb in the engineering bacteria group could be used normally and emit bright red light. The hightest voltage could be up to 2.1V

The results showed that the gene we transferred significantly improved the electricity generation capacity of *S. oneidensis* MR-1, which can actually provide electrical energy for some electrical appliances.

### 4.3 Co-culture system

##### 4.3.1 Co-culture growth

We attempted to combine the BG11 medium required for *Synechocystis*  sp. PCC 6803 culture with the M9 buffer used for *S. oneidensis* MR-1 to generate electricity and configured MBG11 medium to achieve the growth of two kinds of bacteria through the same medium, so as to prepare for the subsequent continuous culture experiment.

We used MBG11 culture medium to culture *Synechocystis*  sp. PCC 6803 and *S. oneidensis* MR-1 respectively, and measured OD values of *Synechocystis*  sp. PCC 6803 culture medium at 730nm and *S. oneidensis* MR-1 culture medium at 600nm. The OD value is measured every 24 hours, and a growth curve is obtained as follows. It can be seen that MBG11 culture can make the normal growth of *Synechocystis*.

However, at the same time, we found that *S. oneidensis* MR-1 could grow at present, but the growth rate was slow. When the bacteria reached the stable stage, the concentration of the bacteria was not high, and the growth situation was far less than that cultured in LB medium. We analyzed that the reason for this result might be the lack of nutrients.

At the same time, we also noticed that when M9 buffer was used to generate electricity for a long time, the bacterial concentration in the device was almost unchanged, indicating that in a low-nutrient environment, *S. oneidensis* MR-1 could only maintain survival and generate electricity but could not grow and multiply.

In response to this situation. We will further improve the medium system in the future, and at the same time increase the concentration of *S. oneidensis* MR-1 in LB medium in advance and add MBG11 medium to produce electricity.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig26.png)
*Fig 26. The growth curve of *Synechocystis*  sp. PCC 6803 and *Synechocystis*  sp. PCC 6803(omcs)in MBG11.Both *Synechocystis*  sp. PCC 6803 and *Synechocystis*  sp. PCC 6803(omcs) can grow at fast speed relatively. Compared to *Synechocystis*  sp. PCC 6803, *Synechocystis*  sp. PCC 6803 (omcs) exhibited a significant improvement in cell concentrationat all stages, with a more stable growth rate. This provides evidence that omcs can also enhance ATP synthesis and promote the photosynthetic efficiency of PS1 in the MBG11 culture environment, resulting in better cell growth.*
![](https://static.igem.wiki/teams/4595/wiki/results/results/fig27.png)
*Fig 27. The growth curve of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) in MBG11.*

##### 4.3.2 Co-culture to produce electricity

We used MBG11 medium to measure the electric production performance of Shiwanella in logarithmic growth phase. The results are as follows, indicating that it can produce electricity normally in MBG11 medium containing lactic acid, and further prove the feasibility of co-culture scheme.

![](https://static.igem.wiki/teams/4595/wiki/results/results/fig28.png)
*Fig 27. The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) in MBG11.*

![arrow-down-square](https://2022.igem.wiki/hust-china/static/arrow-down-square.svg)
![arrow-up-square](https://2022.igem.wiki/hust-china/static/arrow-up-square.svg)

Access Wiki Tools
View Source Code
![]()