iGEM 2022 — Team cuhksz


## Design

Escherichia coli possesses two distinct nitrite reductases. The NirB nitrite reductase is a soluble enzyme that uses NADH as an electron donor to reduce nitrite in the cytoplasm. The NrfA nitrite reductase is a membrane-associated respiratory enzyme that couples to the membrane-associated enzymes via quinones. They are responsible for the six-electron reduction of nitrite to ammonia. NirB and NrfA reductases are two key enzymes in E. coli participating in its nitrate dissimilatory pathway (Figure.1). With this basis, our project aims to elevate the expression of the nitrite reductase gene, and ultimately the protein level.

![1A](https://static.igem.wiki/teams/4459/wiki/engineering/figure-1.png)

To achieve this, the first step is to determine which gene is of our favorite.
By further investigating the relationship of NirB and NrfA reductases by literature review, we found that these two enzymes function in a complementary way. Under lower nitrite concentration
(<1.25mM), nrfA gene is preferentially expressed, serving as the ‘first response’, while the expression of NirB is more significantly induced under higher nitrite concentration (>3.5mM).
This complementary expression indicates that NrfA reductases has a higher affinity to nitrite than NirB reductases (Wang & Gunsalus, 2000).
Also, since our target tested samples are mainly water and food, the implementation of our products asks for a high sensitivity and accuracy, nrfA was chosen to be our target gene.

To amplify the expression of the gene, the most common way is to directly over-express the target gene.
But in our case, the nrfA gene is regulated by nrfABCDEFG operon, implying NrfA reductase is not functioning independently.
In fact, Nrf proteins do cooperate tightly to maintain the function of NrfA reductase. For example the NrfB is the direct electron donor for NrfA, and NrfC plays a role to reduce the oxidized NrfB(Figure.2).
Thus, simply over-express NrfA will only drain out the whole system. But it’s also not ideal to over-express the whole operon due to the redundancy and complexity.
Thus, we thought about utilizing transcriptional factors to boost the whole system. nrfA gene is up-regulated by two TFs, NarL and NarP (Figure.3).
According to Wang & Gunsalus, NarP could regulate the nrfA gene in wider range of nitrite.

Moreover, to make the nitrite detection more specific and accessible, more things should be dealt. If we look again at Figure.3,
FNR is also a transcriptional factor of nrfA gene. In fact, FNR functions to switch many bacteria from aerobic to anaerobic metabolism.
By direct interaction with oxygen, Fe-S complex of FNR is quickly converted to [3Fe-4S] + or a [2Fe-2S]2+, inactivating FNR (Unden et al., 1997).
Thus, we must ensure an anabolic environment to avoid the failure or bias of the detection.
Our first instinct was to directly create an oxygen-free environment by ventilating the culture media with nitrogen.

![2 ](https://static.igem.wiki/teams/4459/wiki/engineering/figure-2.png)

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![3](https://static.igem.wiki/teams/4459/wiki/engineering/figure-3.png)

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## Build

We then had to choose the appropriate plasmid. The pGEM-T-Easy plasmid was finally chosen for its simple structure and a T7 promoter right before the CMS, making the narP gene insertion easier. We also preserved the 5’UTR of the narP gene to help with the transcription and translation.

We then had to choose the appropriate plasmid. pGEM-T-Easy plasmid was finally chosen for its simple structure and a T7 promoter right before the CMS, making the narP gene insertion easier. We also preserved the 5’UTR of the narP gene to help with the transcription and translation. But we worried about the over-expressed NarP protein would probably fall into the inclusion body.
To avoid this, we thought about adding the Thioredoxin Tag to increase the solubility and help with the correct folding of proteins.

![4](https://static.igem.wiki/teams/4459/wiki/engineering/figure-4.png)

## Test

Choosing BL21 as the protein expression system, we transformed the recombinant plasmid using heat shock transformation. Positive colonies could then be selected from the AMP plate for amplification. We further extracted the plasmid and sent it for sequencing to validate the transformation efficiency and avoid the potential risk of gaps or mismatches.

Hooray! The result shows a perfect match between our design and the extracted plasmid, indicating that we had theoretically achieved the goal of our project.

![5](https://static.igem.wiki/teams/4459/wiki/engineering/figure-5.png)

Our team further performed a series of experiments to verify the efficacy of our plasmid. We researched both the transcriptional and protein levels to examine the expression level of narP and NrfA. After several rounds of experiments, we changed the time duration of ventilating nitrogen from two hours to overnight. The validation results are listed as follows:

![6](https://static.igem.wiki/teams/4459/wiki/engineering/figure-6.png)

Figure 6 shows that after transformation, the expression level of narP and narI genes becomes significantly higher. However, for our target protein NrfA, its expression value was undetermined, which implied that the up-regulation effect of narP on NrfA may be blocked.

![7](https://static.igem.wiki/teams/4459/wiki/engineering/figure-7.png)

In this experiment, we succeeded in obtaining the relative enzyme activity. The relative enzyme activities of the four groups were arranged as follows: after transformation anaerobic > after transformation aerobic >> before transformation aerobic > before transformation anaerobic. This result confirmed our transformation’s success, and it had a significant positive effect on enzyme expression. However, the effect of anaerobic conditions was very small, which was not consistent with our expectations.

## Analysis

The experiment results caused our concerns about the inclusion body. Explicitly, we worried that the over-expressed narP protein would fall into the inclusion body. To avoid this, we thought about adding the Thioredoxin Tag to increase the solubility and help improve the correctness of folding.

The low expression of NrfA might result from the failure to create the anaerobic environment. During the experiments, we found it challenging to exclude oxygen and evaluate whether the environment is proper enough for enzyme production. To figure out the underlying mechanism, we investigated and predicted all possible binding sites of FNR on the promoter region of nrfA. According to the results, FNR was predicted to have multiple binding sites, indicating that only an extreme anaerobic condition can induce the activity of this protein.

## Future Design

In further experiments, instead of trying to satisfy the experimental condition,
we decide to work on FNR structure. In complement to this, we will consider to introduce a modified oxygen-insensitive FNR mutant protein by substituting either Cys-23 or Cys-122 with Ser (Bates et al., 1995),
which is presented in Figure.8.

![8](https://static.igem.wiki/teams/4459/wiki/engineering/figure-8.png)

We may also have to deal with one confounder to increase the specificity. Figure 2 shows that nitrate could be converted to nitrite in two ways in low and high nitrate conditions. Figure 6 also reflects an overexpression of genes encoding nitrate reductases NarI. Therefore, our project would then introduce RNAi to eliminate the effect of nitrate.
y.

The traditional RNAi depends on RNAi machinery in eukaryotes, which is not functional in prokaryotes. The prokaryotic method for invader sequences incorporates these fragments into their clusters of regularly interspaced short palindromic repeat (CRISPR) locus. Then the transcript interacts with the Cas protein to form the effector. Among various Cas proteins, the Cas RAMP, or Cmr, complex exclusively targets and cleaves RNA but not DNA. Cmr complex exists in P. furiosus to cut specifically ssRNA only.

![9](https://static.igem.wiki/teams/4459/wiki/engineering/figure-9.png)

Based on this principle, the plasmid can be designed. The whole cmr complex is clone into the plasmid with their original promoter.
Target sequence that is complementary to the target RNA is followed behind with a 5’ tag of conserved sequence ‘5′ AUUGAAAG 3′’ (Hale et al., 2012).

![10](https://static.igem.wiki/teams/4459/wiki/engineering/figure-10.png)

## Reference

Wang, H., & Gunsalus, R. P. (2000). The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. Journal of bacteriology, 182(20), 5813–5822. https://doi.org/10.1128/JB.182.20.5813-5822.2000

Unden, G., & Schirawski, J. (1997). The oxygen-responsive transcriptional regulator FNR of Escherichia coli: the search for signals and reactions. Molecular microbiology, 25(2), 205–210. https://doi.org/10.1046/j.1365-2958.1997.4731841.x

Palmer, I., & Wingfield, P. T. (2004). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Current protocols in protein science, Chapter 6, Unit–6.3. https://doi.org/10.1002/0471140864.ps0603s38

Bates, D. M., Lazazzera, B. A., & Kiley, P. J. (1995). Characterization of FNR\* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR. Journal of bacteriology, 177(14), 3972–3978. https://doi.org/ 10.1128/jb.177.14.3972-3978.1995

Hale, C. R., Majumdar, S., Elmore, J., Pfister, N., Compton, M., Olson, S., Resch, A. M., Glover, C. V., 3rd, Graveley, B. R., Terns, R. M., & Terns, M. P. (2012). Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Molecular cell, 45(3), 292–302. https://doi.org/10.1016/j.molcel.2011.10.02

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Experiments

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Results

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