iGEM 2024 — Team sustechocean

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![顶部图片](https://static.igem.wiki/teams/5397/header/notebook_1.png)

OUTLINE

## Notebook

*Synechococcus sp.* PCC7942

Stage1: Strain Culture(7.1-7.30)

* ### OD750 Measurement

  ### 7.1-7.30

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/1-1-1.png)
* ### OD750 Measurement

  ### 7.1-7.30

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/1-1-2.png)
  Table 2: Growth curve of cyanobacteria Cultured in a light incubator.

Stage2: Strain Transformation(7.17-8.29)

* ### 7.17

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/1-2-1.png)
  DH5α amplification of cscB gene and cscB plasmid extraction.
* ### 7.23

  Plasmid extraction of cscB and transformation into PCC7942.
* ### 8.1

  Measurement of cscB plasmid concentration and sample submission for testing.
* ### 8.29

  SN medium used for wild type, pre-transformation, and post-transformation.

Stage3: Expression Verification(9.7-9.22)

* ![](https://static.igem.wiki/teams/5397/experiment-notebook/1-3-1.png)
  We measured sucrose content from September 7 to 22, showing that transformed
  cyanobacteria had decreased intracellular sucrose and increased supernatant sucrose,
  indicating their ability to secrete sucrose.
* ![](https://static.igem.wiki/teams/5397/experiment-notebook/1-3-2.png)

*S. oneidensis* MR-1

Stage1: Strain Culture(7.9-7.22)

* ### 7.9

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-1-0.png)
  Designed a thiobarbituric acid method for colorimetric detection of formic acid
  concentration, using a microplate reader to measure OD450 absorbance.
* ### 7.14

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-1-1.png)
  Design three electrosynthesis experiments with wild-type*S. oneidensis* MR-1.
* ### 7.22

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-1-2.png)
  Measurement of the resistance of different powered devices.

Stage2: Strain Transformation(7.16-7.24)

* ### 7.16-7.23

  We prepared *S. oneidensis* MR-1 competent cells and attempted electroporation. However, the
  electroporation was unsuccessful.
* ### 7.24

  Through continuous learning and optimization of the procedure, we successfully
  achieved electroporation.

Stage3: Expression Verification(8.1-9.22)

* ### 8.1

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-3-1.png)
  We designed and completed a fourth electrosynthesis experiment, which confirmed the
  successful expression of the designed gene plasmid and improved EEU efficiency.
* ### 8.3

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-3-2.png)
  We designed the 5th and 6th electrosynthesis experiments, which confirmed that the
  addition of 2-HNQ improved EEU efficiency.
* ### 8.4

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-3-3.png)
  We designed the 7th and 8th electrosynthesis experiments, which confirmed that the
  addition of riboflavin improved EEU efficiency.
* ### 8.6

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-3-4.png)
  *S. oneidensis* MR-1 Chelex100 DNA Extraction
* ### 8.21

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/2-3-5.png)
  We completed the 9th and 10th electrosynthesis experiments, which confirmed that the
  addition of riboflavin and rGO improved EEU efficiency.

*Vibrio Natriegens*

Stage1: Strain Culture(7.3-7.16)

* ### 7.3

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-1-1.png)
  Designed four media formulations: 2216E, 2216E + 1.5% NaCl, 2216 + 3% NaCl, 2216E +
  4.5% NaCl for cultivating V.N.
* ### 7.16

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-1-2.png)
  Attempted to Culture *Vibrio natriegens* with sodium formate.

Stage2: Strain Transformation(7.29-9.22)

* ### 7.29

  Cabp-Chbd plasmid extraction.
  First electroporation
* ### 8.2

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-1.png)
  1.Four sets of Cabp-Chbd plasmid extraction：
* ### 8.2

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-2.png)
  Electroporated plasmid.

  100ng plasmid added to 50uL competent V.N.
* ### 8.30

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-3.png)
  V.N.
  electroporation.

  100ng plasmid added to 50uL competent V.N.
* ### 9.3

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-4.png)
  Extraction of CA plasmid from E. coli.
* ### 9.3

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-5.png)
  V.N. electroporation.
* ### 9.5

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-6.png)
  V.N. electroporation. transformation failure.
* ### 9.7

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-7.png)
  V.N. electroporation. transformation failure.
* ### 9.8

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-8.png)
  V.N. electroporation. transformation failure.
* ### 9.16

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-2-9.png)
  V.N. electroporation. transformation failure.

Stage3: Expression Verification(8.26-9.30)

* ### 8.26

  1.Glycerol stock of E.coli Cabp-Chbd plated on resistant medium.
  2. E.coli Cabp-Chbd preserved by streaking.
  3. Induced expression, fluorescence microscopy showed no 580nm excitation light. No
  fluorescence at 550nm.
  Reinduced and wrapped in aluminum foil, attempted fluorescence detection again with
  535B microplate reader.
* ### 8.27

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-1.png)
  Preparation of induction expression system (4 sets).
* ### 9.5

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-2.png)
  0:00: Inoculated E. coli (containing Cabp-Chbd and CA genes), OD600 measured.
* ### 9.5

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-3.png)
  Measured mCherry and EGFP fluorescence intensity (morning)
* ### 9.5

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-4.png)
  Afternoon
  significant change in fluorescence intensity, first induction failed.
* ### 9.16

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-5.png)
  Inoculated V.N., OD600 measured.
* ### 9.27

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-6.png)
  A concentration gradient of IPTG was prepared, and transformed *Vibrio
  natriegens* was inoculated at 22:00. When OD600 reached 0.5, IPTG was added to
  induce fluorescent protein expression, which was observed using a fluorescence
  microscope.
* ### 9.27

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-7.png)
  Upon excitation with red light under the fluorescence microscope, the *Vibrio
  natriegens* transformed with the mCherry gene exhibited a stable red fluorescent
  signal, confirming the successful expression of the plasmid.
  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-8.png)
  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-9.png)
  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-10.png)
* ### 9.30

  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-11.png)
  ![](https://static.igem.wiki/teams/5397/experiment-notebook/3-3-12.png)
  Fluorescence microscopy of *Vibrio natriegens* expressing EGFP showed a strong,
  uniform green fluorescence under 488 nm excitation, with emission around 509 nm,
  confirming correct EGFP expression. This indicates successful transformation and
  effective transcription and translation of the CARPs plasmid in *Vibrio
  natriegens*.

Seawater Analysis Experiment

7.20-7.24

![solar cell](https://static.igem.wiki/teams/5397/experiment-notebook/4-1.png)

Collected water samples from Shenzhen Bay and measured salinity, conductivity, pH,
calcium ion concentration.

7.20-7.24

![solar cell](https://static.igem.wiki/teams/5397/experiment-notebook/4-2.png)

Collected water samples from Shenzhen Bay and measured salinity, conductivity, pH,
calcium ion concentration.

7.20-7.24

![solar cell](https://static.igem.wiki/teams/5397/experiment-notebook/4-3.png)

Collected water samples from Shenzhen Bay and measured salinity, conductivity, pH,
calcium ion concentration.

7.20-7.24

![solar cell](https://static.igem.wiki/teams/5397/experiment-notebook/4-4.png)

Collected water samples from Shenzhen Bay and measured salinity, conductivity, pH,
calcium ion concentration.

7.20-7.24

![solar cell](https://static.igem.wiki/teams/5397/experiment-notebook/4-5.png)

Collected water samples from Shenzhen Bay and measured salinity, conductivity, pH,
calcium ion concentration.

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![顶部图片](https://static.igem.wiki/teams/5397/header/engineering.png)

OUTLINE

![](https://static.igem.wiki/teams/5397/engineering/dbtl/1.png)

[Synechococcus sp.PCC7942](%7B%7Burl_for%28%27pages%27%2Cpage%3D%27engineering%27%29%7D%7D)
[S.oneidensis MR-1](%7B%7Burl_for%28%27pages%27%2Cpage%3D%27soneidensis%27%29%7D%7D)
[Vibrio natriegens](%7B%7Burl_for%28%27pages%27%2Cpage%3D%27vibrio_natriegens%27%29%7D%7D)

## Overview

![](https://static.igem.wiki/teams/5397/experiment-engineering/overview.png)

## Cycle0: Plasmid Design

Design

•
The plasmid introduced into the cyanobacteria was obtained from the ShanghaiTech\_China 2022
team. They
constructed a plasmid containing the Sucrose permease (cscB) gene, which can integrate into the
cyanobacterial genome. We expressed this plasmid in S. elongatus. For more information, please
check the
ShanghaiTech\_China 2022 website at [Engineering Success |
ShanghaiTech\_China -
iGEM 2022](https://2022.igem.wiki/shanghaitech-china/engineering)
![](https://static.igem.wiki/teams/5397/engineering/1/1.png)

## Cycle1: Culturing

Design

•
We decided to cultivate the cyanobacteria using BG11 liquid medium, inoculating the strains at a
ratio of
100:1. The cultures were maintained at room temperature under natural light conditions. The
optical density
at 750 nm (OD750) was measured daily using a spectrophotometer to plot the growth curve.

Build

![](https://static.igem.wiki/teams/5397/engineering/1/2.png)

Room temperature under natural light

Test

the cyanobacteria entered the logarithmic phase on day 15 and reached the stationary phase on
day 27.
![](https://static.igem.wiki/teams/5397/engineering/1/3.png)

Learn

![](https://static.igem.wiki/teams/5397/engineering/1/4.png)

25°C constant illumination incubator (6000 lux) led to a faster growth rate

![](https://static.igem.wiki/teams/5397/engineering/1/5.png)

The constant illumination incubator (6000 lux) led to a faster growth rate

## Cycle2: Stain Transformation

Design

•We reviewed literature to obtain the cscB gene sequence
for sucrose secretion and had a plasmid with regulatory elements synthesized.

Build

•We cultured 1.5 ml of cyanobacteria (OD750 = 0.5),
centrifuged, resuspended in BG11 medium, and added plasmid (≥1 ng/μl). The mixture was incubated
at 30°C for 24 hours.

Test

•After 24 hours, we spread the culture on BG11 agar plates
with antibiotics and incubated until single colonies appeared, confirming successful
transformation.

Learn

•We identified transformed colonies using antibiotic
selection, measured sucrose production, and adjusted conditions to optimize cscB expression,
documenting the process throughout.

## Cycle3: Expression Validation

Design

•We selected engineered PCC7942 strains for cscB gene
expression experiments, planned their cultivation in BG11 medium with ampicillin, and prepared
the necessary equipment and reagents, including a sucrose assay kit.

Build

•Single colonies from antibiotic-resistant plates were
inoculated into BG11 medium with ampicillin and cultured for seven days until OD685 reached 0.5
to ensure sufficient growth and gene expression.

Test

•50 ml cultures of wild-type and engineered PCC7942 were
centrifuged, resuspended in BG11 medium, and sucrose levels were measured every 24 hours for 72
hours using a sucrose assay kit. Intracellular sucrose was also measured with additional
freeze-thawing, ultrasonication, and extraction steps.

Learn

•Data was analyzed to evaluate cscB gene expression and
sucrose production. Based on results, adjustments to the expression system or conditions were
made to improve future experiments.

References

* Atsushi Kouzuma, Molecular mechanisms regulating the catabolic and electrochemical
  activities of Shewanella oneidensis MR-1, Bioscience, Biotechnology, and Biochemistry, Volume
  85, Issue 7, July 2021, Pages 1572-1581, https://doi.org/10.1093/bbb/zbab088
* Kasai, T., et al., Overexpression of the adenylate cyclase gene cyaC facilitates current
  generation by Shewanella oneidensis in bioelectrochemical systems. Bioelectrochemistry, 2019.
  129: p. 100-105.
* Sun, W., et al., Promoting Extracellular Electron Transfer of Shewanella oneidensis MR-1 by
  Optimizing the Periplasmic Cytochrome c Network. Front Microbiol, 2021. 12: p. 727709.
* Li, F., Li, YX., Cao, YX. et al. Modular engineering to increase intracellular NAD(H/+)
  promotes rate of extracellular electron transfer of Shewanella oneidensis. Nat Commun 9, 3637
  (2018). https://doi.org/10.1038/s41467-018-05995-8
* Li, F., Li, Y. X., Cao, Y. X., Wang, L., Liu, C. G., Shi, L., & Song, H. (2018). Modular
  engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer
  of Shewanella oneidensis. Nature communications, 9(1), 3637.
  https://doi.org/10.1038/s41467-018-05995-8
* Cao, Y., Song, M., Li, F., Li, C., Lin, X., Chen, Y., Chen, Y., Xu, J., Ding, Q., & Song, H.
  (2019). A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1. Frontiers in
  microbiology, 10, 410. https://doi.org/10.3389/fmicb.2019.00410
* AHUT\_China iGEM Team. (2018). Part: BBa\_K2547003. Retrieved from
  https://parts.igem.org/Part:BBa\_K2547003
* Cao, Y., Song, M., Li, F., Li, C., Lin, X., Chen, Y., Chen, Y., Xu, J., Ding, Q., & Song, H.
  (2019). A synthetic plasmid toolkit for Shewanella oneidensis MR-1. Frontiers in Microbiology,
  10, 410. https://doi.org/10.3389/fmicb.2019.00410
* Delgado, V. P., Paquete, C. M., Sturm, G., & Gescher, J. (2019). Improvement of the electron
  transfer rate in Shewanella oneidensis MR-1 using a tailored periplasmic protein composition.
  Bioelectrochemistry (Amsterdam, Netherlands), 129, 18-25.
  https://doi.org/10.1016/j.bioelechem.2019.04.022
* Li, F., Li, Y. X., Cao, Y. X., Wang, L., Liu, C. G., Shi, L., & Song, H. (2018). Modular
  engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer
  of Shewanella oneidensis. Nature Communications, 9(1), 3637.
  https://doi.org/10.1038/s41467-018-05995-8
* iGEM06\_MIT. (2008). Part: BBa\_B0029. Retrieved from https://parts.igem.org/Part:BBa\_B0029
* iGEM22\_ShanghaiTech\_China. (2022). Part: BBa\_K4115045. Retrieved from
  https://parts.igem.org/Part:BBa\_K4115045
* SCUT-China iGEM Team. (2023). Part: BBa\_K4621004. Retrieved from
  https://parts.igem.org/Part:BBa\_K4621004
* Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K., & Tanaka, H. (1990). Gene cloning of
  chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia
  chitinase and to the type III homology units of fibronectin. Journal of Bacteriology, 172(1),
  401-409. https://doi.org/10.1128/jb.172.1.401-409.1990
* Weiss, R. (n.d.). Part: BBa\_B0030. Retrieved from https://parts.igem.org/Part:BBa\_B0030

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![顶部图片](https://static.igem.wiki/teams/5397/header/results.png)

OUTLINE

PCC7942

Growth Curve

Two sets of growth curves were obtained.

| Group | Temperature | Illumination |
| --- | --- | --- |
| 1 | Room temperature | Natural illumination |
| 2 | 25℃ | 6000lux |

![](https://static.igem.wiki/teams/5397/experiment-notebook/1-1-1.png)

Figure 1: Growth curve of cyanobacteria cultured under room temperature and natural light conditions.

![](https://static.igem.wiki/teams/5397/experiment-notebook/1-1-2.png)

Table 2: Growth curve of cyanobacteria cultured in a light incubator.

Expression Verification

The data in the figure show that the intracellular sucrose content in transformed cyanobacteria decreased, while the sucrose content in the supernatant increased, indicating that the transformed cyanobacteria are capable of secreting sucrose outside the cell.
![](https://static.igem.wiki/teams/5397/experiment-results/result3.png)

![](https://static.igem.wiki/teams/5397/experiment-results/result4.png)

*Shewanella oneidensis* MR-1

Colony and Growth Rate

Colony:
![](https://static.igem.wiki/teams/5397/experiment-results/result5.jpg)

Figure 1. Colony of *Shewanella oneidensis*> MR-1 on TSA plate

Growth Conditions: 200mL LB Broth in 500mL Erlenmeyer flask, 30 oC, 225 rpm
Every two hours we measured optical density (OD600).
![](https://static.igem.wiki/teams/5397/experiment-notebook/2-1-0.png)

Figure 2. *S. oneidensis* MR-1 Growth Curve

Growth Rate (R) = 0.026 generations/min

Generation Time (G) = 1 / R = 38.40 min

Exploring the Essential Conditions for Electrosynthesis in *Shewanella oneidensis* MR-1

The **wild type strain** was used in this experiment. The different conditions set were whether electricity was applied (if applied, then at 1V) and **whether carbon dioxide was introduced (if introduced, the flow rate was controlled at 60 mL/min)**, while other conditions remained the same. The formate concentration in the device was measured every 2 hours.

| Group No. | Strain | Initial OD600 | Electricity Applied | C02 Flow | Eletron mediator |
| --- | --- | --- | --- | --- | --- |
| 1 | WT | 0.812 | N | N | N |
| 2 | WT | 0.812 | N | Y | N |
| 3 | WT | 0.812 | Y | Y | N |

![](https://static.igem.wiki/teams/5397/experiment-results/result7.png)

Figure 3. Electrosynthesis of formic acid in *S. oneidensis* MR-1 is activated by electricity.

According to data, the group with both electricity and CO2 flow applied showed a surge in the amount of formic acid produced, while the one with only CO2 flow added didn’t change evidently compared to the group without any treatment. It demonstrated that the electrosynthesis system is activated by exogenous electricity instead of CO2, which is consistent with the fact we have already known that this system is the reverse of EET pathway, and electrons are required to drive the synthesis. On the contrary, CO2 itself didn’t affect this process possibly due to low activity of electrosynthesis system. At 6h, the concentration of formic acid lowers, probably because of the effect of massive bacteria colony, leading to a total consumption. From this result we know that electricity is necessary for efficient electrosynthesis of Formic Acid. To produce as much as possible formic acid, CO2, the raw material, should also be added.

Examine the Effect of *Shewanella oneidensis* MR-1 with Overexpression of ccta on Formate Production

| Group No. | Strain | Initial OD600 | Electricity Applied | C02 Flow | Eletron mediator |
| --- | --- | --- | --- | --- | --- |
| ... | ... | ... | ... | ... | ... |
| 4 | Engineered Strain | 0.731 | Y | Y | N |

![](https://static.igem.wiki/teams/5397/experiment-results/result8.png)

Figure 4. Engineered Strain possessed a higher production of formate.

We imported a plasmid containing ccta gene in order to increase its overexpression, therefore contributing to a higher rate of electrosynthesis, whose principle is explained in design. After the transformation, the concentration of formic acid produced by engineered strain is higher than that by WT strain, with the peak difference about 2mM, increased by 23.4%. Note that the original OD of engineered strain was slightly lower than WT, so this increase could be higher. This result indicates that our design is feasible and effective to rise the electrosynthesis rate.

Compare the Effects of Different Electron Mediators

| Group No. | Strain | Initial OD600 | Electricity Applied | C02 Flow | Eletron mediator |
| --- | --- | --- | --- | --- | --- |
| ... | ... | ... | ... | ... | ... |
| 5 | WT | 0.766 | Y | Y | 2-HNQ |
| 6 | Engineered Strain | 0.752 | Y | Y | 2-HNQ |
| 7 | WT | 0.766 | Y | Y | RF |
| 8 | Engineered Strain | 0.752 | Y | Y | RF |
| 9 | WT | 0.784 | Y | Y | RF+rGO |
| 10 | Engineered Strain | 0.773 | Y | Y | RF+rGO |

Figure 5. Electron mediators have effects on electrosynthesis. The production of formic acid after adding electron mediators to (A) WT or (B) engineered strain. The comparison between WT and engineered strain is visualized in (C).

To explore the effects of electron mediators on the electrosynthesis of formic acid, we tested 2-HNQ, RF and RF+rGO in both engineered and WT strain. In both strains, the trend showed a similar pattern: all the mediators improved the amount of formic acid synthesized, which is more obviously shown in WT strain (about 25% increase in average, while in engineered strain, only a 6% increase is observe). The increase is definitely caused by a mediated, faster electron transport. Only subtle effect on engineered strain is likely because electron transfer is not a limiting factor any more thanks to overexpression of ccta. 2-HNQ had smallest influence on the concentration of formate in both strains. RF+rGO reached a highest increase for WT (33.7%), while this effect is lower than RF in engineered strain (RF+rGO 5.8%, only RF 7.3%). Overall, engineered strain were still capable of more active electrosynthesis, which proved our successful design again. As a result, it is advisable to use electron mediators, especially RF or RF+rGO to increase electrosynthesis.

Vibrio Natrigens

V.N. Culturing

Designed four media formulations for cultivating V.N.
OD measured every two hours
![](https://static.igem.wiki/teams/5397/experiment-results/result10.png)

Analysis: High salinity in the medium, replaced with a different medium based on literature research.

We designed three groups of M9 minimal media with different concentrations of sodium formate as the carbon source and sucrose as the auxiliary carbon source to culture Vibrio natriegens. As shown in the figure, the results indicate that:

1. Vibrio natriegens can utilize sodium formate as a carbon source for survival.
2. in the groups with sucrose as an auxiliary carbon source, Vibrio natriegens exhibited rapid growth.

![](https://static.igem.wiki/teams/5397/experiment-results/result11.png)

Fluorescence Detection

Upon excitation with red light under the fluorescence microscope, the Vibrio natriegens transformed with the mCherry gene exhibited a stable red fluorescent signal, confirming the successful expression of the CA plasmid.
![](https://static.igem.wiki/teams/5397/experiment-results/result12.jpg)
![](https://static.igem.wiki/teams/5397/experiment-results/result13.jpg)

Through fluorescence microscopy, the detection of Vibrio natriegens samples expressing EGFP showed a significant green fluorescent signal under appropriate excitation at around 488 nm.The observed green fluorescence confirms the successful transformation and expression of the CARPs plasmid, demonstrating that the exogenous gene was effectively transcribed and translated in Vibrio natriegens.
![](https://static.igem.wiki/teams/5397/experiment-results/result14.jpg)

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