iGEM 2024 — Team nanjing-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

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# Notebook

notebook.pdf

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

- [1. Overview](#s1)

* [1.1 Pre-experiments](#s11)
* [1.2 Pre-experimental conclusion](#s12)

- [2. Cycle One](#s2)

* [2.1 Design](#s21)
* [2.2 Build](#s22)
* [2.3 Test](#s23)
* [2.4 Learn](#s24)

- [3. Cycle Two](#s3)

* [3.1 Design](#s31)
* [3.2 Build](#s32)
* [3.3 Test](#s33)
* [3.4 Learn](#s34)

- [4. Dry Lab](#s4)

* [4.1 Design](#s41)
* [4.2 Mathematical modeling results from the Dry Lab](#s42)
* [4.3 Conclusion](#s43)

- [5. Cycle Three](#s5)

* [5.1 Design](#s51)
* [5.2 Build](#s52)
* [5.3 Test](#s53)

- [6. Cycle Four](#s6)

* [6.1 Design](#s61)
* [6.2 Build](#s62)
* [6.3 Test](#s63)
* [6.4 Learn](#s64)

- [7. Summary of the Engineering Section](#s7)

- [References](#s8)

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# Engineering

# 1. Overview

Our goal is to explore the relationship between phosphorus-related metabolism and electricity-producing functions in *Shewanella* onediensis MR-1(*Shewanella*). We hope to use synthetic biology to modify an engineered *Shewanella*. that can act both as a phosphorus uptake in wastewater and produce electricity.

### 1.1 Pre-experiments

In order to find out in advance what happens to the electrogenic function and the phosphorus polymerisation function in wild-type *Shewanella* and how they are related to each other, we carried out a series of pre-experiments, measured some basic parameters and obtained a number of useful regularity conclusions

#### 1.1.1 Detection of phosphorus uptake function

We firstly measured how much phosphorus was absorbed by *Shewanella* after incubation in phosphorus-containing medium for several hours, and calculated the amount of phosphorus absorbed by *Shewanella* using the difference between free phosphorus before and after incubation in the medium, and found that *Shewanella* absorbed phosphorus significantly in the medium at 12h compared with that at 0h, and then the phosphorus in the medium tended to stabilise.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig1.png)

Figure1: Illustration of the net uptake of free phosphorus in the medium by wild type Shewanella as the incubation time progresses.

#### 1.1.2 Electroproduction assay

We then also determined the intensity of the electricity produced by *Shewanella* after incubation (M9 medium) for a number of hours, where we measured the magnitude of the current produced by the organisms as detected in a specific container to indicate the organism's ability to produce electricity. We found that the intensity of the current produced by *Shewanella* showed a significant increase from 1h to 20h, followed by a gradual decrease. In contrast, the cultured *Shewanella* with phosphorus removed demonstrated a much smaller capacity for electricity production than the former.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig2.png)

Figure2: Illustration of the progressive determination of the magnitude of the current produced by Shewanella as the incubation time is extended.Pi-full: 1.62 mg-P/mL;Pi-free: 0 my-P/mL.

### 1.2 Pre-experimental conclusion

Based on the above results, we guessed the following conjecture: the electroproduction ability of *Shewanella* and phosphorus uptake are positively correlated, and the process of accumulation of phosphorus is conducive to the realization of the electroproduction function of *Shewanella*. With the above experimental phenomenon becomes the theoretical basis for our subsequent modification of iGEM cycles.

# 2. Cycle One

We hope to start by improving the ability of *Shewanella* to accumulate polyphosphate.According to the results of our pre-experiments, theoretically, *Shewanella* should show a rise in electricity production capacity.

### 2.1 Design

After our literature research, we initially understood that the center of phosphorus metabolism in *Shewanella* is PolyP[[1]](#ref1), so it is natural to think that by introducing PolyP synthase in *Shewanella*, the ability of *Shewanella* to accumulate polyphosphate can be increased, i.e., the efficiency of *Shewanella* electricity production is improved. And after our research, we selected one of the most common PolyP synthases in *Shewanella* - [PPK1](https://parts.igem.org/Part%3ABBa_K5034213) [[2]](#ref2).

### 2.2 Build

We chose [pBBR1MCS-terminator](https://parts.igem.org/Part%3ABBa_K5034201), the most commonly used plasmid in *Shewanella*, and used it as a backbone to construct the pBBR1MCS-terminator plasmid [B0034-PPK1](https://parts.igem.org/Part%3ABBa_K5034216) with *PPK1*. Named SPK1 (“**S**” for our experimental subject: “*Shewanella*” and “**PK1**” for *PPK1*).

![beaker](https://static.igem.wiki/teams/5034/engineering/fig3.png)
Figure3: Plasmid construction idea diagram for introducing PPK1 into Shewanella

### 2.3 Test

#### 2.3.1 Phosphorus uptake function test after introducing *PPK1*

However, despite our performance on accumulating phosphorus, the electricity generation effect is not satisfactory. We used the difference in free phosphorus (Pi) in the Luria-Bertani medium before and after incubation as an indicator of phosphorus accumulation by the bacteria and obtained the following graph:

![beaker](https://static.igem.wiki/teams/5034/engineering/fig4.png)

Figure4: Comparison of the polyphosphate capacity of both WT and SPK1

We found that the polyphosphate of our engineered bacteria was significantly elevated.

#### 2.3.2 Phosphorus electroproduction assay after introduction of *PPK1*

We have measured the electroproduction capacity of the bacterium according to the experimental protocol in the [Experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) section, and the results show a significant decrease in the electroproduction capacity. This seems to be contrary to the results of our pre-experiment.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig5.png)

Figure5: Comparison of WT and SPK1 power production capacity

### 2.4 Learn

In cycle1 we seem to have come across the exact opposite conclusion from the pre-experiment. Although it is quite understandable that our engineered bacterium did have a significant increase in its ability to accumulate phosphorus, the unsatisfactory results of the power production we hypothesized that the engineered bacterium may have accumulated too much phosphorus and synthesized too much PolyP after the introduction of PPK1, which resulted in an imbalance of the environment within the bacterium.

The morphology of SPK1 that we did later found that the engineered bacterium did show an increase in cell membrane folds and disruption of normal intracellular results (Fig7-8), suggesting that the excessive production of PolyP would have a significant negative impact on the bacterium.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig6.png)

Figure6: Electron microscope observation of the morphology of the Shewanella of WT(upper panel) and SPK1(lower panel). It can be seen that compared to WT, SPK1 has a large number of non-smooth folds on the cell surface.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig7.png)

Figure7: Fluorescence microscopy was used to observe the morphology of the cells of WT and SPK1. The body of SPK1 showed an abnormal shape with a thin middle section and two thick sections.

# 3. Cycle Two

### 3.1 Design

To attenuate the expression of PPK1 in *Shewanella*, we designed three RBSs upstream of the *PPK1* gene, whose translational intensities were named SPK1 to 3 in descending order ([B0031-PPK1](https://parts.igem.org/Part%3ABBa_K5034214), [B0032-PPK1](https://parts.igem.org/Part%3ABBa_K5034215), [B0034-PPK1](https://parts.igem.org/Part%3ABBa_K5034216)). Accumulation capacity of elemental phosphorus, and the electroproduction capacity of *Shewanella* was also determined along similar lines as before.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig8.png)

Figure8: Designed PPK1 plasmids with three different RBS, named SPK1 to 3.

### 3.2 Build

We verified by DNA electrophoresis that we have successfully constructed three sets of plasmids for *PPK1*.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig9.png)

Figure9: Colony PCR of bacterial liquid to determine whether the engineered bacteria have imported the target gene fragments properly.

The results of protein electrophoresis showed that our constructed plasmid did express PPK1 in the bacterium, and the bands deepened in color with the elevated expression. This indicates that the designed expression system with three different RBSs worked properly.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig10.png)

Figure10: Protein electrophoresis to detect whether PPK1 protein is expressed in engineered bacteria.

### 3.3 Test

As before, we determined the accumulation capacity of elemental phosphorus and electricity production capacity of the engineered bacteria as shown in the following figures.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig11.png)

Figure11: Determination of accumulation capacity of elemental phosphorus of three engineered bacteria.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig12.png)

Figure12: Determination of the power-producing capacity of three engineered bacteria.

As shown in the figure, we found that as expected, the capacity to accumulate polyphosphate did decrease after weakening the expression of PPK1. However, the power-producing ability still did not meet our requirements, and although it was much better than SPK1, it still could not be separated from WT. This suggests that importing PPK1 to synthesize PolyP in *Shewanella* may not be able to enhance the power-producing ability of *Shewanella*.

### 3.4 Learn

In fact, the cycle is clearly successful. From the perspective of phosphorus accumulation capacity and electricity production capacity, the result obtained in the first cycle is that the phosphorus accumulation capacity increases and the electricity production capacity decreases significantly. Through the refinement of our second cycle, we have found a way to significantly increase the phosphorus accumulation capacity of *Shewanella* with little or no change (or slight increase) in its ability to produce electricity.

All of the above experimental results suggest that the ability of *Shewanella* to accumulate polyphosphate may not simply be positively correlated with the ability to produce electricity. After our discussion, the option of introducing an enzyme that synthesizes PolyP was considered to be largely ruled out. Focusing instead on PolyP-related phosphorus metabolism, another class of enzymes caught our attention ———— PolyP hydrolases. The following speculations were given:

* After the introduction of PolyP hydrolase, the metabolic level of the engineered bacteria was elevated, which promoted the conversion of polymerised phosphorus into the rest of the small organic phosphorus molecules (e.g., ATP, NADP+, etc.), and elevated the metabolic level of the bacterium, which resulted in a reduction in the amount of PolyP, and thus, according to the principle of the equilibrium of the chemical reaction, the bacterium's ability to polymerise phosphorus should be increased accordingly.

In this speculation, we simultaneously discussed with the students of our dry lab group, who tried to analyze the rationality of the scheme of introducing hydrolytic enzymes in the pre-experimental analysis related to mathematical modeling.

# 4. Dry Lab

### 4.1 Design

After the wet lab students first conducted literature research, the dry lab students were provided with several groups of PolyP hydrolases that we might use, namely:

| Abbreviations | Full name | enzyme | enzyme with backbone(pBBR1MCS-terminator) |
| --- | --- | --- | --- |
| PPX | Exo-polyphosphatase[[3]](#ref3) | [PPX](https://parts.igem.org/Part%3ABBa_K5034210) | [pBBR1MCS-terminator-*PPX*](https://parts.igem.org/Part%3ABBa_K5034229) |
| PPK2 | Polyphosphate kinase 2[[5]](#ref5) | [PPK2](https://parts.igem.org/Part%3ABBa_K5034205) | [pBBR1MCS-terminator-*PPK2*](https://parts.igem.org/Part%3ABBa_K5034227) |
| PAP | AMP phosphotransferase[[6]](#ref6) | [PAP](https://parts.igem.org/Part%3ABBa_K5034212) | [pBBR1MCS-terminator-*PAP*](https://parts.igem.org/Part%3ABBa_K5034219) |
| PPN1 | Polyphosphatase[[3]](#ref3) | [PPN1](https://parts.igem.org/Part%3ABBa_K5034209) | [pBBR1MCS-terminator-*PPN1*](https://parts.igem.org/Part%3ABBa_K5034231) |
| NADK | Poly(P)/ATP-NAD kinase[[7]](#ref7) | [NADK](https://parts.igem.org/Part%3ABBa_K5034206) | [pBBR1MCS-terminator-*NADK*](https://parts.igem.org/Part%3ABBa_K5034225) |

![beaker](https://static.igem.wiki/teams/5034/engineering/fig13.png)

Figure13: A list of functions of several enzymes that hydrolyze PolyP.

### 4.2 Mathematical modeling results from the Dry Lab

After checking in with the dry lab group of students, each of our 2 reliable dry lab members brought us happy news.

#### 4.2.1 Mathematical modeling of phosphorus metabolism network prediction in *Shewanella*

PPK2 and NADK transfer the energy of the high-energy phosphate bonds in PolyP into GTP and NADP+(which is further converted to NADPH), and the products are more active, while PPX, PPN1, and PAP hydrolyze PolyP into lower activity products (i.e., Pi, oligophosphate, and ADP). According to the law of energy conservation, we believe that the energy utilization efficiency of *PPK2* and *NADK* is higher than that of *PPX*, *PPN1*, and *PAP*, which is in line with our goal of increasing the electroproduction capacity of Shewanella. Therefore, we modeled the introduction of *PPK2* and *NADK*.

The modeling results of our dry lab experiments showed that after the introduction of *PPK2* or *NADK*, the ATP content of *Shewanella* increased compared to WT, indicating an increase in the metabolic level. Meanwhile, both the electroproduction capacity and phosphorus accumulation capacity of *Shewanella* increased, indicating that *Shewanella* with high metabolic level possesses better electroproduction and phosphorus accumulation capacity.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig14.png)

Figure14: Predictions of the strength of PPK2 and NADK introductions, respectively, for the generation of currents at specified times in Shewanella.

#### 4.2.2 AI-based modeling to mine genes positively correlated to the electroproducing capacity of Schizosaccharomyces pombe

We compared the prediction confidence of related genes and the mean confidence of all genes and top 50 confidence value. In the NADK group, for example, a substantial increase of confidence in NADH synthesis related, membrane phosphorus trnasportation related and lactate metabolism related genes is shown, in comparison to mean value.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig15.png)

Figure15: prediction confidence of experiment group NADK.

### 4.3 Conclusion

The conclusions of the members of the dry lab experimental group suggest that our shift from the introduction of PolyP synthase to the introduction of PolyP hydrolase strategy may indeed be theoretically beneficial in compensating for the lack of *Shewanella*'s electroproduction efficiency.

# 5. Cycle Three

Our students in the dry lab group gave us confidence, we then constructed separate lines for each of the groups of enzymes we were studying, and tested them for both polyphosphate and electricity production on the line after introduction.

### 5.1 Design

We constructed several structurally similar PolyP hydrolase-containing plasmids as shown below.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig16.png)

Figure16: Designed plasmid elements containing five PolyP hydrolases.

### 5.2 Build

We performed colony PCR and the results showed that our plasmid construction was successful.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig17.png)

Figure17: Colony PCR of bacterial liquid to determine whether the engineered bacteria have imported the target gene fragments properly.

### 5.3 Test

After testing, we found that the electroproduction capacity of *Shewanella* imported with SPPK2 or SNADK was significantly increased compared to the wild type, and we also measured the ATP and NADH/NAD+ content in both, and found that the ATP and NADH level of *Shewanella* imported with the two enzymes mentioned above was also significantly increased.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig18.png)

Figure18: Determination of phosphorus polymerization and electroproduction capacity of a series of polyP hydrolases introduced.

In this round of experiments we found that the SPPK2 and SNADK groups of hydrolases had a nice increase in power production efficiency, and we subsequently also measured two substances indicative of metabolic strength, ATP and NADH, in *Shewanella*, and found that both of these substances were relatively elevated in these same two groups compared to the wild-type, hinting at a potentially positive correlation between metabolic strength and power production levels.

The above experiments effectively show that the protocol of introducing PolyP hydrolase is indeed effective in improving the efficiency of electricity production in *Shewanella*, but the ability of polyphosphate still needs to be improved.

# 6. Cycle Four

Since the above scheme of importing a single PolyP hydrolase worked, we would like to proceed with continued optimization. Since we are now left with two enzymes from the screening ———— SPPK2 and SNADK. Our current idea is to import them both into *Shewanella*, but this is a step that we took before proceeding with the actual construction in collaboration with our dry lab group students. we collaborated on [mathematical modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) of the strategy for introducing the two enzymes to try to predict the outcome ahead of time.

![beaker](https://static.igem.wiki/teams/5034/modelling.png)

The experiment result of ODE models

### 6.1 Design

The results of the dry lab experiment further gave us confidence that we were going to take up the idea of co-expressing the two enzymes, and we attempted to construct a new plasmid and introduce it into *Shewanella*.

### 6.2 Build

Similarly to the previous protocol, we tandemly linked both *PPK2* and *NADK* on top of one plasmid ([pBBR1MCS-terminator-B0034-PPK2-B0034-PPK1](https://parts.igem.org/Part%3ABBa_K5034223))and introduced them into *Shewanella*.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig24.png)

Figure19: Designed plasmid elements containing both NADK and PPK2 hydrolases in tandem.

After colony PCR, we verified that the dual enzyme plasmid construction was successful.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig20.png)

Figure20: Colony PCR results of the two-enzyme strategy.

### 6.3 Test

We also tested the polyphosphate and electroproduction capacities, and were surprised to find that the co-expression of the two PolyP hydrolases effectively enhanced the polyphosphate and electroproduction capacities of *Shewanella*.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig21.png)

Figure21: Determination of the electroproduction and phosphorus accumulation capacities of Shewanella after co-expression of two hydrolytic enzymes.

Comparing the results with WT, it can be seen that after the introduction of dual hydrolase, the accumulation capacity of the engineered bacteria and phosphorus and the electricity production capacity are greatly improved (among them, the electricity production capacity is improved by more than 2 times).

![beaker](https://static.igem.wiki/teams/5034/engineering/fig22.png)

Figure22: Electrical methods for detecting the power-producing capacity of engineered bacteria.Cyclic voltammetry (CV) curve (left panel), Linear sweep voltammetry (LSV) curve (center panel) and Output power density curve (right panel).

The results of the half-cell experiments indicated an elevated electron transfer activity, with currents of 137.4 ± 16.34 µA/cm² for the SPPK2 strain, 134.56 ± 17.01 µA/cm² for the SNADK strain, and 164.2 ± 17.64 µA/cm² for the SPPK2-NADK strain. This resulted in an overall enhancement of up to 20% in power production compared to the previous cycle. The overall increase in power production compared to the previous cycle was approximately 20%.

![beaker](https://static.igem.wiki/teams/5034/engineering/fig23.png)

Figure23: Determination of both NADH/NAD+ and ATP levels in wild-type and Shewanella co-expressing PPK2 and NADK.

At the same time, we also measured two key substances indicative of metabolic intensity in the cell, NADH and ATP, in response to the LEARN section of our previous CYCLE3, and the results were also as expected, with a significant rise in the levels of both of these high-energy phosphate compounds in *Shewanella*, a result which further confirms that boosting the metabolic strength of *Shewanella* is favourable to the enhancement of its electrogenic capacity.

### 6.4 Learn

These two enzymes, as enzymes synthesizing ATP and NADP, have a very important role in the metabolic activity of *Shewanella*. The reason that the two enhance the ability of *Shewanella* to produce electricity is most likely due to the increased metabolic strength of *Shewanella* .

This cycle was able to achieve promising results thanks to our collaboration with the modeling work of the students in the dry lab group and our research with the literature. Obviously, some of the guidance derived from the modeling of the dry lab was able to inform the work of our wet lab group to a certain extent.

# 7. Summary of the Engineering Section

We went through 4 cycles, starting from the beginning with the introduction of PolyP synthase to the introduction of dual hydrolase, which had a lot of ups and downs and misgivings. However, in the end, we were able to obtain an engineered *Shewanella* with a 2-fold increase in power production compared to the wild type: an engineered *Shewanella* with both **PPK2 and NADK** enzymes, which has both good power production efficiency and the ability to accumulate phosphorus from the environment, which also gave us a sense of the charm of synthetic biology.

During the rounds of thinking and arguing about the topic, we gradually realised that synthetic biology is a discipline that requires rigorous logic and diversified thinking, which is indispensable for the careful construction of the four cycles of the project and the patient communication between the members of the dry and wet lab groups. We gained a wealth of theoretical knowledge, practical research experience and team communication skills, which is very meaningful.

# References

[1]: Shi, Tingyu, & Wang, Huailin, & Xie, Jianping. (2011)Progress in Polyphosphate and Related Metabolizing Enzymes[j].Progress in Physiological Sciences,(03),23-29.

[2]: Wang X, Wang X, Hui K, Wei W, Zhang W, Miao A, Xiao L, Yang L. Highly Effective Polyphosphate Synthesis, Phosphate Removal, and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-Copy Plasmid Strategy. Environ Sci Technol. 2018 Jan 2;52(1):214-222. doi: 10.1021/acs.est.7b04532. Epub 2017 Dec 13. PMID: 29190088.

[3]: Lichko LP, Kulakovskaya TV, Kulaev IS. Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Yeast. 2006 Jul 30;23(10):735-40. doi: 10.1002/yea.1391. PMID: 16862600.

[4]: Zhang H, Ishige K, Kornberg A. A polyphosphate kinase (PPK2) widely conserved in bacteria. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16678-83. doi: 10.1073/pnas.262655199. Epub 2002 Dec 16. PMID: 12486232; PMCID: PMC139203.

[5]: Neville N, Roberge N, Jia Z. Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence. Int J Mol Sci. 2022 Jan 8;23(2):670. doi: 10.3390/ijms23020670. PMID: 35054854; PMCID: PMC8776046.

[6]: Itoh H, Shiba T. Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. J Bacteriol. 2004 Aug;186(15):5178-81. doi: 10.1128/JB.186.15.5178-5181.2004. PMID: 15262957; PMCID: PMC451603.

[7]: Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. doi: 10.1016/S1389-1723(04)00302-0. PMID: 16233726.

{% endblock %}

{% 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 %}

- [1. Extract the plasmid](#s1)

- [2. Plasmid double digestion](#s2)

- [3. Gel DNA Purification](#s3)

- [4. PCR for inserts](#s4)

- [5. Recombination](#s5)

- [6. Transformation](#s6)

- [7. Colony PCR](#s7)

- [8. Preparation of Competent *Shewanella onediensis* MR. 1](#s8)

- [9. Measurement of the *Shewanella*](#s9)

- [10. Agarose Gel Electrophoresis](#s10)

- [11. Polyacrylamide gel electrophoresis](#s18)

- [12. Pi content detection](#s11)

- [13. PolyP content detection](#s12)

- [14. ATP content detection](#s13)

- [15. NADH content detection](#s14)

- [16. Scanning electron microscopy and transmission electron microscope characterization](#s15)

- [17. Half-cell experiment](#s16)

- [18. Full-cell experiment](#s17)

![Menu ](https://static.igem.wiki/teams/5034/wiki-images/h-guide-image-small.png)
![Logo](https://static.igem.wiki/teams/5034/wiki-images/team-logo.png)

# Experiments

# 1. Extract the plasmid

Vazyme | FastPure Plasmid Mini Kit (DC201)

### 1.1 Materials

RNase A, Buffer P1, Buffer P2, Buffer P3, Buffer PW1, Buffer PW2, Elution Buffer, FastPure DNA Mini Columns, Collection Tubes 2 ml

### 1.2 Protocol

1. Transfer $1-5 \mathrm{mL}$ of overnight ( $ 12 - 16 h$ ) culture to a centrifuge tube, and centrifuge at $10,000 \mathrm{rpm}$ $(11,500 \times \mathrm{g})$ for $1 min$ . Discard the culture medium and place the tube inverted on a blotting paper to drain the liquid.
2. Add $250 \mu L$ of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.
3. Add $250 \mu L$ of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube $8-10$ times to completely lyse the cells.
4. Add $350 \mu L$ of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently $8 - 10$ times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $10 min$ .
5. Place FastPure DNA Mini Columns into a Collection Tube $2 mL$ . Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette. Taking care not to disturb the precipitates. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30-60 \mathrm{sec}$. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
6. Add $500 \mu L$ of Buffer PW1 to the FastPure DNA Mini Columns. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30 - 60 sec$ . Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.- Add $600 \mu L$ of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $30-60 \mathrm{sec}$. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
   - Repeat Step 7.
   - Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at $12,000\ \mathrm{rpm}(13,400 \times \mathrm{g})$ for $1 min$ to completely remove the residual wash buffer.
   - Place the FastPure DNA Mini Columns in a new sterile $1.5 mL$ centrifuge tube. Add $30-100 \mu L$ of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for $2 min$ , and centrifuge at $12,000 \mathrm{rpm}(13,400 \times \mathrm{g})$ for $1 min$ to elute the DNA.
   - Discard the FastPure DNA Mini Columns and store the extracted DNA at $-20^{\circ}$ C to prevent degradation.

# 2. Plasmid double digestion

### 2.1 Materials

QuickCut${ }^{\text {TM }}$ $EcoR I$ , QuickCut${ }^{\text {TM }}$ $Not I$ , $10 \times$ QuickCut Green Buffer, Plasmid, ddH₂O

### 2.2 Protocol

1. Take $1-2\mu g$ of plasmid and prepare the reaction system on ice as follows:

   | Reagent name | Reagent dosage |
   | --- | --- |
   | QuickCut${ }^{\text {TM }}$ $EcoR I$ | $1 \mu L$ |
   | QuickCut${ }^{\text {TM }}$ $Not I$ | $1 \mu L$ |
   | 10×QuickCut Green Buffer | $5 \mu L$ |
   | Plasmid | $1\mu L -2 \mu L$ |
   | ddH₂O | to$50 \mu L$ |
2. React in $37^{\circ} \mathrm{C}$ for $1-2 \mathrm{~h}$.

# 3. Gel DNA Purification

### 3.1 Materials

Tubes, Buffer GDP, FastPure DNA Mini Columns-G adsorption column, Buffer GW, Elution Buffer.

### 3.2 Protocol

1. After the DNA electrophoresis, quickly cut the gel containing the target DNA fragment under the UV lamp, and it is recommended to use a paper towel to absorb the gel surface solution and try to remove excess gel. Weigh the gel (remove the weight of empty tubes), $100 mg$ gel is equivalent to $100 \mu L$ volume, as gel volume.
2. Add an equal volume of Buffer GDP. Take a water bath at $50 \sim 55^{\circ} \mathrm{C}$ for $7-10 \mathrm{~min}$, and adjust the time appropriately according to the size of the gel to ensure the glue block dissolves completely. Invert and mix 2 times during the water bath to accelerate the sol.
3. Collect droplets from the tube wall by brief centrifugation. Place the FastPure DNA Mini Columns-G adsorption column in Collection Tubes. Transfer $700 \mu L$ of sol solution to the adsorption column in a $2 mL$ collection tube and centrifuge at 12,000 rpm $(13,800 \times \mathrm{g})$ for $30 \ to \ 60 \ sec$ .
4. Discard the filtrate and place the adsorption column in the collection tube. Add $300 \mu L$ of Buffer GDP to the adsorption column. Let stand for 1 min. $12,000 \mathrm{rpm}(13,800 \times \mathrm{g})$ centrifugation for $30-60 \ sec$.
5. Discard the filtrate and place the adsorption column in the collection tube. Add $700 \mu L$ of Buffer GW (absolute ethanol added) to the adsorption column. $12,000 rpm$ ( $13,800 \times \mathrm{g}$ ) centrifugation for $30-60 \ sec$.
6. Repeat step 5 .
7. Discard the filtrate and put the adsorption column back into the collection tube. Centrifuge at $12,000 \mathrm{rpm}$ $(13,800 \times \mathrm{g})$ for 2 min.
8. Place the adsorption column in a $1.5 mL$ sterilized centrifuge tube, add $20-30 \mu L$ of Elution Buffer to the center of the adsorption column, and leave for $2 min$. Centrifuge at $12,000 rpm$ ( $13,800 \times \mathrm{g})$ for $1 min$. Discard the adsorption column and store the DNA at $-20^{\circ} \mathrm{C}$.

# 4. PCR for inserts

### 4.1 Materials

Template, $2 \times$ Phanta, Primer F, Primer R, ddH₂O

### 4.2 Protocol

1. Prepare the reaction solution according to the following table:

   | Reagent name | Reagent dosage |
   | --- | --- |
   | Template1 | $400 ng$ |
   | $2 \times$ Phanta | $25.0 \mu L$ |
   | Primer F | $2.0 \mu L$ |
   | Primer R | $2.0 \mu L$ |
   | ddH₂O | to$50.0 \mu L$ |
2. Run the PCR reaction:

   | Temperature | Duration | Number of cycles |
   | --- | --- | --- |
   | $98^{\circ} \mathrm{C}$ | $30 s$ | 1 cycle |
   | $98^{\circ} \mathrm{C}$ | $10 s$ | 35 cycles |
   | $55^{\circ} \mathrm{C}$ | $5 s$ | 35 cycles |
   | $72^{\circ} \mathrm{C}$ | $5 \mathrm{~s} / \mathrm{kb}$ | 35 cycles |
   | $72^{\circ} \mathrm{C}$ | $1 min$ | 1 cycle |

# 5. Recombination

Vazyme | ClonExpress Multis One Step Cloning Kit (C113)

### 5.1 Materials

Linearized vector, Insert, $5 \times$ CE MultiS Buffer, Exnase MultiS, ddH₂O

### 5.2 Protocol

1. Dilute the vector and insert fragments at an appropriate ratio before preparing the recombination reaction system, and the amount of each component is not less than $1 \mu L$.
2. Prepare the following reaction on ice:

   | Reagent name | Reagent dosage |
   | --- | --- |
   | Linearized Vector | $X \mu L$ |
   | Insert | $Y\_{1}-Y\_{n} \mu L$ (Volume of 1-n insertion fragments) |
   | $5 \times$ CE MultiS Buffer | $4 \mu \mathrm{I}$ |
   | Exnase MultiS | $2 \mu L$ |
   | ddH₂O | to$20 \mu L$ |
3. Gently pipette up and down for several times to mix thoroughly (don't vortex!). Centrifuge to collect the reaction solution to the bottom of the tube.
4. Incubate at $37^{\circ} \mathrm{C}$ for $30 min$ and immediately chill the tube at $4^{\circ} \mathrm{C}$ or on ice.

# 6. Transformation

1. Thaw the competent cells on ice (e.g., DH5α Competent Cell, Vazyme #C502).
2. Pipette $10 \mu L$ of the recombination products to $100 \mu L$ of competent cells, flick the tube wall to mix thoroughly (don't vortex!), and then place the tube on ice for $30 min$ .
3. Heat shock at $42^{\circ} \mathrm{C}$ water bath for $45 sec$ and then immediately place on ice for $2 - 3 min$.
4. Add $900 \mu L$ of LB liquid medium (without antibiotics). Then, shake at $37^{\circ} \mathrm{C}$ for $1 h$ at $200-250$ rpm.
5. Preheat the corresponding resistant LB solid medium plates in a $37^{\circ} \mathrm{C}$ incubator.
6. Centrifuge the culture at $5,000 \mathrm{rpm}(2,400 \times \mathrm{g})$ for $5 min$ , discard $900 \mu L$ of supernatant. Then, use the remaining medium to suspend the bacteria and use a sterile bent glass rod to gently spread on the plate which contains the appropriate selection antibiotic.
7. Incubate at $37^{\circ} \mathrm{C}$ for $12-16 \mathrm{~h}$.

# 7. Colony PCR

Vazyme | $2 \times$ Rapid Taq Master Mix (P222)

### 7.1 Materials

$2 \times$ Rapid Taq Master Mix, Primer 1, Primer 2, colonies, ddH₂O

### 7.2 Protocol

1. Pick a small number of colonies with a pipette tip and shake well in $30\mu L $ of ddH₂O.
2. Place in $95^{\circ} \mathrm{C}$ and heat for $10 min$, centrifuge at 8000 g for 3 min, and keep the supernatant for next step.
3. Prepare the reaction system

| Reagent name | Reagent dosage |
| --- | --- |
| ddH₂O | to$20 \mu L$ |
| $2 \times$ Rapid Taq Master Mix | $10.0 \mu \mathrm{l}$ |
| Primer$1(10 \mu \mathrm{M})$ | $1.0 \mu \mathrm{l}$ |
| Primer$2(10 \mu \mathrm{M})$ | $1.0 \mu \mathrm{l}$ |
| Template DNA | $2.0 \mu \mathrm{l}$ |

# 8. Preparation of Competent *Shewanella oneidensis* MR. 1

### 8.1 Materials

*Shewanella oneidensis*  MR.1, Sterile pre-chilled water, Sterile pre-chilled $10 \%$ glycerol

### 8.2 Protocol

1. Inoculate overnight cultured *Shewanella* into freshly cultured $5 mL$ LB medium and incubate for $3-3.5 \mathrm{~h}$ to $\mathrm{OD}=0.4-0.6$
2. Centrifuge at $2,000 \mathrm{rpm}$ for $15 min$ and wash with $200 mM$ sucrose.
3. Repeat the previous step $3$ times and retain the Bacterial precipitation.
4. Resuspend the cells with $1 mL$ of sucrose and aliquot $100 \mu L$ for the preparation of competent cells.
5. Add $400 ng$ of plasmid, electrotransfer into competent cells with $1.5 kV$ , and transform at $30{ }^{\circ} \mathrm{C}$ until the transformant grows.

# 9. Measurement of the *Shewanella oneidensis* MR.1

### 9.1 Materials

Bacterial culture medium, Cellulase

### 9.2 Protocol

1. Take 3 sterilized ep tube, labeled 1, 2, and 3. Add $2.5 mL$ of LB culture medium and $500 \mu L$ bacteria respectively.
2. Set the initial OD value of the bacterial solution to $0.02$ . The volume of bacterial culture medium to be added is calculated.
3. Put it into a shaker for culture, and measure the OD value every $3 \ hours$ .

# 10. Agarose Gel Electrophoresis

### 10.1 Materials

GelRed nucleic acid dyes, DL2000 DNA Marker, DL5000 DNA Marker, PCR products, Double digestion products

### 10.2 Protocol

1. Add $1.2g$ of agarose and $80 mL$ of $1 \times$ TAE buffer to a Erlenmeyer flask, and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave. Using a low to medium setting, set the timer for a minimum of $5 min$, stopping the microwave oven every $30 sec$ and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to $60^{\circ} \mathrm{C}$ before adding $2 \mu L$ GelRed nucleic acid dyes.
5. Place the comb into the $15 \times 7 \mathrm{~cm}$ slot of the tray and pour the molten agarose into the gel tray.
6. Allow $30-40 min$ for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath $2\ to\ 6 mm$ of $1 \times$ TAE buffer.
9. Load the samples and the marker.
10. Close the lid of gel tank and apply a voltage of $75 V$.
11. turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

# 11. Polyacrylamide gel electrophoresis

### 11.1 Materials

Stacker A, Stacker B, Resolver A (12%), Resolver B, APS, Protein.

### 11.2 Protocol

1. Mix each component upside down 6-8 times before use.
2. Preparation of the lower gel: take the same volume of Resolver A 2.7 $mL$ and Resolver B 2.7 $mL$ each, and mix well.
3. Preparation of upper gel: take the same volume of Stacker A 0.75 $mL$ and Stacker B 0.75 $mL$, each, and mix well.
4. Add $60.0 \mu L$ of APS to the mixing solution in step 2 and mix well. immediately. Inject into the gelatinizing glass plate so that the liquid level is about 1.5 cm from the upper edge of the short glass plate.
5. Add $15.0 \mu L$ of APS to the mixing solution in step 3 and mix well immediately. Without waiting for the lower gel to solidify, gently inject the mixed solution into the gel-making glass plate and gently insert the comb teeth.
6. After the gel has solidified (about 15min at room temperature), the comb teeth can be removed and used for electrophoresis. The recommended electrophoresis voltage is 150V, and the electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge.
7. Then when the sample is loaded, add 5 μL of 10-180 kDa protein label.
8. 5ug protein loading buffer is loaded in the protein sample we detect.
9. Heated at 95 degrees for 10min, and $10.0 \mu L$ protein is loaded after 150kv electrophoresis for 1h, and then decolorized.

# 12. Pi content detection

### 12.1 Materials

Molybdate, Ascorbic acid, ddH2O

### 12.2 Protocol

1. Prepare molybdate solution at a concentration of $130 \mathrm{~g} / \mathrm{L}$ and ascorbic acid solution at a concentration of $10 \%(w / v)$.
2. the supernatant to be measured was mixed with molybdate solution, ascorbic acid solution and water at a volume ratio of $1: 2: 1: 49$
3. Detect the absorbance value at 710 nm was determined after standing for 10 min .

# 13. PolyP content detection

### 13.1 Materials

Bacteria, Lysate, Hepes buffer, DAPI

### 13.2 Protocol

1. $10^{8}$ bacteria cultured overnight $(O D=1.0)$ were washed once with $20 mM$ Hepes buffer solution.
2. Centrifuged at $12,000 rpm$ for $5 min$ , then resuspended with $1 mL$ of Hepes buffer.
3. It is heated at $95{ }^{\circ} \mathrm{C}$ for $10 min$ , and then placed on ice to cool.
4. Take $10 uL$ of lysate and add it to the mixture of $900 uL$ of Hepes buffer and $100 uL$ of $100 mM$ DAPI solution to mix well. Mix well every $5 min$.
5. Repeat the operation twice.
6. Take $200uL$ of the mixture to detect the fluorescence density. Conditions are set to excite at $415 nm$ and emission at $550 nm$.

# 14. ATP content detection

Enhanced ATP Assay Kit

### 14.1 Materials

ATP detection working solution, ATP detection reagent diluent

### 14.2 Protocol

1. $10^{8}$ bacteria $(O D=1.0$ ) cultured overnight are taken and centrifuged at $12,000 rpm$ for $5 min$.
2. Add $200\mu L$ of lysate for follow-up operations. After lysis, centrifuge at $12,000 \mathrm{~g}$ at $4^{\circ} \mathrm{C}$ for 5 min, take the supernatant.
3. Prepare an appropriate amount of ATP testing solution according to the ratio of $100 \mu L$ of ATP detection solution required for each sample or standard.
4. Add $100 \mu L$ of ATP detection solution to the test hole or test tube. Leave at room temperature for $3-5 min$.
5. Add $20 \mu L$ of sample or standard to the test well or test tube, quickly mix well with a micropipette, at least $2 sec$ apart, and measure the RLU value with a chemiluminescence instrument.

# 15. NADH content detection

NAD+ /NADH Assay Kit with WST-8

### 15.1 Materials

NADH preparation liquid, NAD+s>/NADH extract, Reaction buffer, Chromogenic solution

### 15.2 Protocol

1. $10^{8}$ bacteria $(O D=1.0$ ) cultured overnight were also taken and centrifuged at $12,000 rpm$ for $5 min$ .
2. Add $200 uL$ of lysate for follow-up operations. After lysis, centrifuge at $12,000 \times \mathrm{~g}$ at $4^{\circ} \mathrm{C}$ for $5 min$ and take the supernatant,
3. Pipette $50-100 \mu L$ of the sample to be tested in a centrifuge tube and heat for $30 min$ on $60^{\circ}$ C water bath or PCR instrument to break down NAD⁺. If insoluble matter is produced after heating, centrifuge at $10,000 \mathrm{~g}$ for $5 min$ at room temperature or $4^{\circ} \mathrm{C}$ and pipette $20 \mu L$ of the supernatant as the sample to be tested into a 96-well plate.
4. Configure the following systems:

   | Regant name | Blank | Standard | Sample |
   | --- | --- | --- | --- |
   | Samples to be tested | - | $20 \mu \mathrm{l}$ | $20 \mu \mathrm{l}$ |
   | NAD+/NADH extract | $20 \mu \mathrm{l}$ | - | - |
   | Alcohol dehydrogenase working solution | $90 \mu \mathrm{l}$ | $90 \mu \mathrm{l}$ | $90 \mu \mathrm{l}$ |
5. Incubate at $37^{\circ} \mathrm{C}$ for $10 min$ in the dark.
6. Mix the chromogenic solution appropriately, then add $10 \mu L$ of chromogenic solution to each well, mix well, and incubate at $37^{\circ} \mathrm{C}$ for $30 min$ in the dark, at this time orange-yellow formazan will be formed.

Measure the absorbance at $450 nm$ .

# 16. Scanning electron microscopy and transmission electron microscope characterization

### 16.1 Materials

Glutaraldehyde, Ethanol

### 16.2 Protocol

1. Cells were collected by centrifugation for $5 min$ at $12,000 \times \mathrm{rpm}$ and then fixed with $5 \%$ glutaraldehyde at $4^{\circ} \mathrm{C}$ for $12 h$ .
2. Samples were dehydrated in increasing concentrations of ethanol $(25 \%, 50 \%, 75 \%, 95 \%$, and $100 \%$ ) for scanning electron microscopy measurements (SU8020, Japan) or transmission electron microscope (FEI Tecnai G2 F30, USA).

# 17. Half-cell experiment

### 17.1 Materials

Amperotentiostat (CHI1040C, Zhenhua, China), $3 \mathrm{M}\ \mathrm{KCl} \ \mathrm{Ag} / \mathrm{AgCl}$ electrode, Carbon cloth, M9 buffer, Sodium lactate and Luria-Bertani culture

### 17.2 Protocol

1. A single-chamber electrochemical reactor ( $28 mL$ working volume) MEC system consisting of three electrodes in the Half-Cell Laboratory.
2. Prepare the electrode solution. The working electrode is carbon cloth, the reference electrode is a $3 \mathrm{M}\ \mathrm{KCl} \ \mathrm{Ag} / \mathrm{AgCl}$ electrode and the counter electrode is a graphite rod. The electrolytes were M9 buffer, $20 mM$ sodium lactate and $5 \% \mathrm{LB}$, and different concentrations of AgNO₃.
3. The maximum output current was recorded with an amperotentiostat (CHI1040C, Zhenhua, China) by current-time (I-t) measurement ( $200 mV$ on the reference electrode).

# 18. Full-cell experiment

### 18.1 Materials

Two-chamber electrochemical reactor,sodium lactate, KHPO₄, KH₂PO₄, M9 minimal medium, proton exchange membrane, Carbon cloth

### 18.2 Protocol

1. The MFC system of a two-chamber electrochemical reactor ( $140 mL$ working volume) with electrodes connected via a $2 \mathrm{k} \Omega$ external resistor is used to record the output voltage. Carbon cloth of $1 \times 2 \mathrm{~cm}^{2}, 2.5 \times 3.5 \mathrm{~cm}^{2}$ and proton exchange membrane (Nafion 117, United States DuPont) were used as electrodes and separators, respectively.
2. Prepare the electrode solution. The cathode solution is $50 mM$ potassium ferricyanide, $50 mM$ K₂HPO₄, and 50 mM KH₂PO₄ solutions. The anode solution is M9 minimal medium, $20 mM$ sodium lactate, and $5 \%$ LB (Luria-Bertani) medium.
3. Turnover cyclic voltammetry at $1 \mathrm{mV} / \mathrm{s}$ scan rate. Test polarization discharge curves and output power density curves at $0.1 \mathrm{mV} / \mathrm{s}$.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

- [1. Cycle1: Inducing enzymes polyphosphate kinase 1 (PPK1)](#s1)

* [1.1 Pi Concentration and Electricity Generation Capability Testing](#s11)
* [1.2 Analysis of the Factors Contributing to the Reduced Electricity Generation Capability of SPK1](#s12)

- [2. Cycle2: Engineered SPPK1 Strains with Different RBS Designs](#s2)

* [2.1 Plasmid Construction and Transformation](#s21)
* [2.2 Pi Concentration and Electricity Generation Capability Testing](#s22)
* [2.3 The Growth Status of the Engineered Strains](#s23)

- [3. Cycle3: Engineered Strains with Different Hydrolytic Enzyme](#s3)

* [3.1 Plasmid Construction and Transformation](#s31)
* [3.2 Pi Concentration and Electricity Generation Capability Testing](#s32)
* [3.3 Detection of Cellular Metabolic Activity](#s33)

- [4. Cycle4: Combination of PPK2 and NADK enzymes](#s4)

* [4.1 Performance of SPPK2-NADK strain](#s41)
* [4.2 Pi Removal Efficiency Verification](#s42)
* [4.3 Other Electrochemical Parameters Measurement](#s43)

- [5. Supplementary Figures](#s5)
- [6. Reference](#s6)

![Menu ](https://static.igem.wiki/teams/5034/wiki-images/h-guide-image-small.png)
![Logo](https://static.igem.wiki/teams/5034/wiki-images/team-logo.png)

# Pre-experiment results

#### 1. Changes in Inorganic Phosphate During Electricity Generation

We culture *Shewanella* in standard Luria-Bertani medium and measure the inorganic phosphate (Pi) concentration in the medium at 0h, 12h, 24h, and 36h. The results show a decrease in phosphorus concentration in the medium during the electricity generation process (Fig. 1), suggesting that the bacteria require Pi during electrocatalysis.

![beaker](https://static.igem.wiki/teams/5034/results/figure1.png)

Figure 1: Pi concentration in the medium during the cultivation of *Shewanella*.

#### 2. Effect of Pi on Electricity Generation

Next, to investigate the effect of Pi concentration on electron transfer and electricity generation, we culture *Shewanella* in Pi-full and Pi-free medium. Throughout the culture process, we continuously monitor the output current. It turns out that the output current in the Pi-free environment is only 40.8 ± 4.74 µA/cm², which is 60.65% lower than the output current in the Pi-full environment (103.69 ± 8.88 µA/cm²) (Fig. 2), indicating that Pi is crucial for electron transfer.

![beaker](https://static.igem.wiki/teams/5034/results/figure2.png)

Figure 2:Output current-time curve of *Shewanella* under Pi-rich and Pi-free conditions.

# 1. Cycle1: Inducing enzymes polyphosphate kinase 1 (PPK1)

### 1.1 Pi Concentration and Electricity Generation Capability Testing

Due to pre-experiment findings indicating that *Shewanella* requires Pi for electron transfer, we introduce PPK1 enzyme into *Shewanella*. The main function of PPK1 is to polymerize Pi into polyP, thereby promoting polyphosphate accumulation. We connect this gene to the ribosome binding site(RBS) [Registry code: [BBa-B0034](https://parts.igem.org/Part%3ABBa_B0034)] to enhance *PPK1* gene expression. We refer to this engineered strain as SPK1.

Detection results show that SPK1’s polyphosphate production capability is four times greater than that of the wild-type strain(WT), reaching 139.6 mg-P/L. However, the current density is only 23% of that of the WT, measuring just 23.6 µA cm² (Fig. 3). This level of electricity production efficiency is unsatisfactory, prompting us to analyze the reasons for SPK1's weak electricity generation capacity.

![beaker](https://static.igem.wiki/teams/5034/results/figure3.png)

Figure3: Pi concentration and current density in WT and SPK1 strains

### 1.2 Analysis of the Factors Contributing to the Reduced Electricity Generation Capability of SPK1

To investigate the factors of SPK1's reduced electricity generation capability, we examine SPK1 strain using Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). SEM images show that SPK1 bacteria exhibit a less smooth morphology compared to WT (Fig. 4).

![beaker](https://static.igem.wiki/teams/5034/results/figure4.png)

Figure4: SEM images of WT and SPK1 strains.

TEM results indicate that SPK1 bacteria appear to be "cut in half" (Fig. 5). A possible reason is that the high expression of *PPK1* may lead to an accumulation of excessive negative charges within the bacterial cells.

![beaker](https://static.igem.wiki/teams/5034/results/figure5.png)

Figure5: TEM images of WT and SPK1 strains.

In summary, morphological characterization of the bacteria shows that strain with high Pi concentration exhibit abnormal morphology, suggesting that pre-conditioning bacteria to a high polyphosphate state is detrimental to rapid electron transfer.

# 2. Cycle2: Engineered SPPK1 Strains with Different RBS Designs

### 2.1 Plasmid Construction and Transformation

Due to the high polyphosphate state being unfavorable for rapid electron transfer, we explore whether adjusting the strength of the RBS to modify *PPK1* expression can restore electricity production capacity.

We construct three kinds of plasmids with varying strengths of RBS linked to the gene encoding *PPK1* and transform them into wild-type *Shewanella*. The RBS serial numbers used are BBa-B0031, BBa-B0032, and BBa-B0034, with BBa-B0031 having the lowest strength and BBa-B0034 having the highest strength. The resulting strains are named SPK3, SPK2, and SPK1, respectively (Fig. 6).

![beaker](https://static.igem.wiki/teams/5034/results/fig6-new.png)

Figure6: Schematic diagram of PPK1 plasmids construction.

We then perform a series of experiments to verify the successful construction and expression of the plasmids in *Shewanella*. DNA agarose gel electrophoresis results show that we obtain three kinds of plasmids with different RBS sequences, each approximately 2.1 kb in size (Fig. 7).

![beaker](https://static.igem.wiki/teams/5034/results/figure7.png)

Figure7: DNA agarose gel electrophoresis results of three PPK1 fragments

Next, we transform the plasmids into wild-type *Shewanella*, express it, and perform colony PCR and protein extraction for SDS-PAGE. The results show that *PPK1* is successfully introduced into *Shewanella* for replication (Fig. 8).

![beaker](https://static.igem.wiki/teams/5034/results/figure8.png)

Figure8: Colony PCR results of SPK1, SPK2 and SPK3.

As the strength of the RBS increases, the expression level of the PPK1 protein (approximately 70 kD) also increases (Fig. 9).

![beaker](https://static.igem.wiki/teams/5034/results/figure9.png)

Figure9: SDS-PAGE results of proteins from WT, SPK1, SPK2, and SPK3 strains.

### 2.2 Pi Concentration and Electricity Generation Capability Testing

As we expected, as the strength of the RBS decreases, the electrochemical activity of *Shewanella* significantly increases, while polyphosphate capacity decreases. The electricity production capacity of SPK3 is approximately 351.5% higher than that of SPK1. However, the polyphosphate capacity of SPK3 is only about 54.0% lower than that of SPK1, which is not as critical when considering the issue of weaker electricity generation(Fig. 10). Nonetheless, we continue to explore better synthetic biology approaches to further optimize both electricity generation and polyphosphate accumulation capabilities.

![beaker](https://static.igem.wiki/teams/5034/results/figure10.png)

Figure10: Pi concentration and current density in WT, SPK1, SPK2, and SPK3 strains.

### 2.3 The Growth Status of the Engineered Strains

To investigate the growth status of SPK1, SPK2, and SPK3, we culture them in LB medium for 15 hours, measuring the OD600 every three hours. A higher OD600 value indicates a greater number of bacteria. The results show that the bacteria numbers of SPK3, SPK2, and SPK1 are significantly lower than that of the WT strain, with SPK1 exhibiting the least number, indicating that they are in an unfavorable growth state(Fig. 11). This aligns with our previous SEM and TEM results (Fig. 4-5), collectively suggesting that preconditioning bacteria to a high polyphosphate state is detrimental to their growth.

![beaker](https://static.igem.wiki/teams/5034/results/figure11.png)

Figure11: Growth curve of WT, SPK3, SPK2 and SPK1 strains.

# 3. Cycle3: Engineered Strains with Different Hydrolytic Enzyme

### 3.1 Plasmid Construction and Transformation

We hypothesize that regulating the polymerization of phosphates in bacteria may enable control over their electron transfer efficiency. Since PolyP mainly participates in the polymerization and hydrolysis of inorganic phosphate, along with predictions from [dry experimental models](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27ai-model%27%29%7D%7D), our subsequent synthetic biology strategy is to construct engineered strains based on five polyphosphate-metabolizing enzymes(PPX, PPK2, NADK, PAP, PPN1). The five constructed strains are named SPPX, SPPK2, SNADK, SPAP, and SPPN1.

Similarly, we conduct a series of experiments to verify the successful construction of our plasmids and their introduction into *Shewanella*. The results of DNA agarose gel electrophoresis show that we obtain gene fragments for each hydrolase, ranging in size from 1.0 to 2.0 kb. The bands at the bottom of the *PPN1* and *PAP* lanes are primer dimers, which do not interfere with our experiments(Fig. 12).

![beaker](https://static.igem.wiki/teams/5034/results/figure12.png)

Figure12: DNA agarose gel electrophoresis results of gene fragments of five hydrolytic enzymes.

Next, we transform the plasmids into wild-type *Shewanella*, express them, and perform colony PCR. The results also indicate that the genes of each enzyme are successfully introduced into *Shewanella* for replication (Fig. 13).

![beaker](https://static.igem.wiki/teams/5034/results/figure13.png)

Figure13: Colony PCR results of SPPK2, SPPX, SPAP, SPPN1, SNADK.

### 3.2 Pi Concentration and Electricity Generation Capability Testing

After culturing in standard LB medium for 12 hours, we measure the remaining Pi concentration in the medium to indirectly determine the polyphosphate levels of each group. The results indicate that the SPPK2 and SNADK strains have higher polyphosphate accumulation efficiency. Meanwhile, half-cell experiments show increased electron transfer activity, with the SPPK2 strain exhibiting a current density of 137.4 ± 16.34 µA/cm² and the SNADK strain showing 134.56 ± 17.01 µA/cm² (Fig. 14).

![beaker](https://static.igem.wiki/teams/5034/results/figure14.png)

Figure14: Pi concentration and current density after introducing different hydrolytic enzymes.

### 3.3 Detection of Cellular Metabolic Activity

The intracellular ATP content and NADH/NAD+ level in the SPPK2 and SNADK strains are higher than WT strain(Fig. 15), indicating active intracellular metabolism. Therefore, we believe that the increase in electron transfer activity is due to the elevated intracellular metabolic levels of the engineered strains.

![beaker](https://static.igem.wiki/teams/5034/results/figure15.png)

Figure15: Intracellular ATP content and NADH/NAD+ level of Shewanella strains after introducing different hydrolytic enzymes and WT *Shewanella*.

# 4. Cycle4: Combination of PPK2 and NADK enzymes

### 4.1 Performance of SPPK2-NADK strain

Since both PPK2 and NADK exhibit good performance individually, we attempt to combine these two hydrolases in series to see if there will be improved results. Since the *PPK2* gene is approximately 1.1 kb and the *NADK* gene is about 1.0 kb, the *PPK2-NADK* construct should be approximately 2.1 kb. The colony PCR results show a band at about 2.1 kb, confirming that we successfully introduce the plasmid containing *PPK2-NADK* into *Shewanella* (Fig. 16).

![beaker](https://static.igem.wiki/teams/5034/results/figure16.png)

Figure16: Colony PCR results of SPPK2-NADK strain.

Half-cell experiments show increased electron transfer activity, with the current density of the SPPK2-NADK strain measuring 164.2 ± 17.64 µA/cm², approximately 30 µA/cm² higher than that of the SPPK2 and SNADK strains (Fig. 17).

![beaker](https://static.igem.wiki/teams/5034/results/figure17.png)

Figure17: Half-cell experiment results showing electron transfer rates in WT and SPPK2-NADK strains.

With the introduction of PPK2-NADK, the phosphorus accumulation capacity of the SPPK2-NADK strain was also significantly improved.(Fig. 18)

![beaker](https://static.igem.wiki/teams/5034/results/fig24.png)

Figure18: Pi accumulation capacity in WT and SPPK2-NADK strains.

Similarly, the intracellular ATP content and NADH/NAD+ level of the SPPK2-NADK strain are significantly higher, reaching 0.28 µmol/10⁸ cells and 1.79, which are 3.59 times and 5.42 times that of the WT strain (Fig. 19).

![beaker](https://static.igem.wiki/teams/5034/results/figure18.png)

Figure19: Intracellular ATP and NADH/NAD+content of WT strain and SPPK2-NADK strain

### 4.2 Pi Removal Efficiency Verification

The experimental data above indicate that the SPPK2-NADK engineered strain has a significant advantage in electricity production compared to other strains. Given that our goal is to construct an engineered strain that can efficiently catalyze electricity generation while also achieving polyphosphate accumulation,we used M9 cultural medium, which has a higher phosphorus content, in order to explore its application prospects. We then examine the Pi levels at 12, 24, and 36 hours. The results show that the phosphate removal rate increases by 36.8–48.5% during the electron transfer process (Fig. 20).

![beaker](https://static.igem.wiki/teams/5034/results/figure22.png)

Figure20: Pi concentration detection in WT and SPPK2-NADK strains

This demonstrates that the engineered strain SPPK2-NADK successfully facilitates both phosphate removal and electrocatalysis simultaneously.

### 4.3 Other Electrochemical Parameters Measurement

To explore the specific electrochemical performance of the SPPK2-NADK strain, we conduct the following experiments:

Firstly, we use cyclic voltammetry (CV) and linear sweep voltammetry (LSV) techniques for testing. The CV curve shows higher redox activity in the SPPK2-NADK strain (Fig. 21);

![beaker](https://static.igem.wiki/teams/5034/results/figure19.png)

Figure21: Cyclic voltammetry (CV) curves of WT and SPPK2-NADK strains

the LSV curve indicates lower internal resistance in the MFC cells of the SPPK2-NADK strain (Fig. 22).

![beaker](https://static.igem.wiki/teams/5034/results/figure20.png)

Figure22: Linear sweep voltammetry (LSV) curves of WT and SPPK2-NADK strains

Next, we measure the relative output power. The power density results show that the SPPK2-NADK strain has a maximum output power of 243.77 ± 25.2 mW/m², which is 2.32 times higher than the WT strain’s output power density (105.06 ± 11.72 mW/m²) (Fig. 23).

![beaker](https://static.igem.wiki/teams/5034/results/figure21.png)

Figure23: Output power density curves of WT and SPPK2-NADK strains

These results indicate that the electrochemical performance of the SPPK2-NADK strain is significantly better than that of the WT strain.

# 5. Supplementary Figures

![beaker](https://static.igem.wiki/teams/5034/results/fig23.jpg)

Figure 24: The backbone of plasmid after double enzyme digestion

We constructed a plasmid using a double enzyme digestion method, where the cleavage site is EcoR I and Not I. It is about 5000 bp.

# 6. Reference

[1]: Zhang H, Ishige K, Kornberg A. A polyphosphate kinase (PPK2) widely conserved in bacteria. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16678-83. doi: 10.1073/pnas.262655199. Epub 2002 Dec 16. PMID: 12486232; PMCID: PMC139203.

[2]: Mori S, Yamasaki M, Maruyama Y, Momma K, Kawai S, Hashimoto W, Mikami B, Murata K. Crystallographic studies of Mycobacterium tuberculosis polyphosphate/ATP-NAD kinase complexed with NAD. J Biosci Bioeng. 2004;98(5):391-3. doi: 10.1016/S1389-1723(04)00302-0. PMID: 16233726.

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