Recalling our experiment, we engineered BL21 and made an E. coli strain with enhanced nitrite-digesting ability. However, observing several inconsistencies between our expectations and reality, we want to investigate the causes further. Meanwhile, we additionally design more detail to make our design more rigorous and complete. The focuses of our plan mainly involve:

1. Verifying the existence of inclusion body:

After introducing the plasmid containing the narP gene into bacteria, we observed a significant rise in the expression level of NarP from SDS-PAGE. However, the nitrite-digesting ability of the strain was still below our expectations, indicating an unsatisfactory inducing function of NarP to nrfA. In searching for possible reasons, we suspect that the over-expressed NarP protein would fall into the inclusion body, in which the target protein is enclosed by a membrane and cannot function normally.

One way to examine the existence of the inclusion body is to check the insoluble pallet since proteins in the inclusion body do not dissolve in detergent. After centrifugation and detergent, protein in the inclusion body can be solubilized by guanidine·HCl. The extracted unfolded protein can then be folded and run the SDS-PAGE following the protocol (Palmer & Wingfield, 2004). To avoid this, we thought about adding the Thioredoxin Tag to increase the solubility and help the correct folding of proteins.

If the NarP is proven to fall into the inclusion body, we will add a thioredoxin tag to the plasmid, which can make NarP more soluble and assists the correct folding of the protein.

In our experiment design, we set anaerobic groups to promote the expression of NrfA since the Fe-S complex within FNR would be rapidly oxidized under oxygen, blocking FNR from the enhancer region.

We initially decided to create an anaerobic environment; therefore, we ventilated the culture media with nitrogen overnight to remove the oxygen as thoroughly as possible. It could be inferred from our results that the anaerobic condition did make some improvement, but the effect still does not match the expression level of NarP. We concluded that ventilating nitrogen might not be efficient enough to achieve strict anaerobic since it is already long enough to ventilate the culture overnight. Thus, we considered culturing the bacteria within a confined chamber, where oxygen is removed by chemical reagents, or filling the chamber with inert gases. We also change our minds from excluding oxygen to inhibiting the function of the oxygen-responsive protein. Specifically, we intend to introduce a modified oxygen-insensitive FNR mutant protein by substituting either Cys-23 or Cys-122 with Ser, which is predicted to release the expression of nrfA from anaerobic limitation.

Figure 1 Secondary structure of RyhB predicted by RNA fold

RNAi will play an important role in our future experiments to exclude inference and amply the expression of our target protein.

The traditional RNAi depends on RNAi machinery in eukaryotes and thus is not functional in prokaryotes. The prokaryotic method to deal with invader sequences is to incorporate these fragments in to 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. Thus, plasmid can be designed based on this. 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′’.

Figure 2

Figure 3

3. Effect of nitrate reductase:

During our literature review, we found that several nitrate reductases, including NarI and NarZ are also involved in the regulation pathway of NarP. Specifically, nitrate reductase could reduce nitrate to nitrite, introducing a confounder in our detection, and thus should be eliminated. However, due to the time limit, we can only construct the strain and ensure its ability with the three validation methods mentioned before.

In the future, we will pay closer attention to the expression level of NarI and NarZ to see whether their expression levels are affected by the over-expressed NarP. If the answer is yes, we will design correlated siRNA sequences targeting the nitrate reductases and perform RNAi. To tackle this issue, we plan to inhibit the expression of these nitrate reductases by applying RNAi, which can specifically induce the degradation of target mRNA and silence their corresponding genes.

In previous studies, we found that the expression level of NirB in E. coli was consistently relatively low, and the expression level of NirB in E. coli transformed with narP gene was not significantly higher than that before the transformation. We were very puzzled by this, and after repeated tests to determine that there was no severe error in the experimental operation, we thought that some regulatory mechanism endogenous to E. coli reduced the expression level of NirB. We believe that bacterial small RNA (sRNA) may play an important role in this. sRNA regulators act through base pairing with RNAs, usually modulating the translation and stability of mRNAs. The majority of these sRNAs regulate responses to changes in environmental conditions.

To prove our hypothesis, we searched and performed a blast-based phylogenetic analysis in sRNATarBase (A comprehensive database of bacterial sRNA targets verified by experiments). Finally, we found that an sRNA named ryhB can bind to mRNA encoding nirB, thereby inhibiting translation, then reduce the expression level of nirB. In order to ensure the accuracy of this result, we also consulted a large number of pieces of literature and finally found that there are experimental results that the binding of ryhB to nirB mRNA can significantly inhibit the expression of the modified protein.

Based on the above, we plan to try the expression of ryhB in E. coli in future experiments by including but not limited to CRISPR-CAS knockout or down-regulation of the gene encoding ryhB and binding it with molecules such as peptide nucleic acids that can specifically bind to ryhB level, and thus the expression level of nirB.

Figure 4 Secondary structure of RyhB predicted by RNA fold

First, the sensitivity of our potentiostat has to be further improved to meet our detection requirements. The current detection limit of our potentiostat could reach the order of 10-3, however, 10-4 to 10-5 would be ideal. Therefore, to meet the requirement to apply our electrochemical station to wider application fields, more effort has to be devoted to improving its sensitivity performance. Second, the cell lysates and contents may have interfered with our nitrite detection, as although the result shows a positive linear relationship between the nitrite concentration in the sample solution and the peak value of the current response, the range of response variation is too small, indicating that our sensor needs further improvement in the detection sensitivity. In our future study, more work will be done to avoid the interference in our detection process.

6. Human Practices

In response to our expectation for this project, providing a real-time and easy-operative nitrite monitor that is reachable for every common people, integrated human practices are scheduled for a better application prospect. Specifically speaking, we will communicate with customers, cafeterias, environmental monitoring, and medical testing companies, which correspondingly represent our product's three main target customers to investigate the market demand. After we finish manufacturing, we will invite them to test our nitrisensor and collect their feedback for refinement. We will also visit testing companies and manufacturers for a more profound understanding of nitrite detection and cooperate with them to produce the nitrisensor in a large-scale.