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.

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.

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], 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 ^[2].

2.2 Build

We chose pBBR1MCS-terminator, the most commonly used plasmid in Shewanella, and used it as a backbone to construct the pBBR1MCS-terminator plasmid B0034-PPK1 with PPK1. Named SPK1 (“S” for our experimental subject: “Shewanella” and “PK1” for PPK1).

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:

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 section, and the results show a significant decrease in the electroproduction capacity. This seems to be contrary to the results of our pre-experiment.

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.

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, B0032-PPK1, B0034-PPK1). Accumulation capacity of elemental phosphorus, and the electroproduction capacity of Shewanella was also determined along similar lines as before.

3.2 Build

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

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.

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.

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:

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.

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.

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.

5.2 Build

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

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.

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 of the strategy for introducing the two enzymes to try to predict the outcome ahead of time.

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)and introduced them into Shewanella.

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

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.

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).

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%.

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

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