View details: BBa_K4459001 , BBa_K4459002 , BBa_K4459003
pGEM-T-Easy-narP
The addition of nitrite elevates the expression of nitrite reductase via either the NarL or NarP. However, NarL and NarP play a slightly different role. After antitheses of selectively expressing certain transcription factors, the experiment group with NarP overexpressing and the expression of NarL be suppressed, behaves powerfully activating efficacy, indicating a predominate influence of NarP over NarL. Therefore, this project chooses to up-regulate the expression level of the NarP to overexpress the nrfA operon it regulates. In theory, introducing our designed plasmid could significantly improve the expression level of NarP. When the E. coli adapts itself to the anaerobic condition, with the binding of FNR, overexpressed NarP could then up-regulate the expression of nrfA, equipping the E. coli with a more robust capability to sense and deal nitrite it encounters.
Oxygen-insensitive FNR
To make nitrite detection more specific and more accessible, we have to deal with two more things. We notice that FNR is also a transcriptional factor of the nrfA gene. In fact, FNR is acting as a switch for 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, one obvious limitation of our project is that we have to ensure an anabolic environment, which may increase the failure or bias of the detection. To solve this we try to introduce a modified oxygen-insensitive FNR mutant protein by substituting either Cys-23 or Cys-122 was substituted for Ser and substitution at position 154.
After the site mutation, the sequence of the protein is shown below: >Modified oxygen-insensitive FNR mutant protein MIPEKRIIRRIQSGGCAIHCQDCSISQLCIPFTLNEHELDQLDNIIERKKPIQKGQTLFKAGDELKSLYAIRSGTIKSYTITEQGDEQITGFHLAGDLVGFDAIGSGHHPSFAQALETSMVSEIPFETLDDLSGKMPNLRQQMMRLMSGEIKGAQDMILLLSKKNAEERLAAFIYNLSRRFAQRGFSPREFRLTMTRGDIGNYLGLTVETISRLLGRFQKSGMLAVKGKYITIENNDALAQLAGHTRNVA
To obtain 3D structure of modified FNR, we use alphafold 2.1.0 (The ColabFold AlphaFold2 notebook by Sergey Ovchinnikov, Milot Mirdita and Martin Steinegger, which uses an API hosted at the Södinglab based on the MMseqs2 server (Mirdita et al. 2019, Bioinformatics) for the multiple sequence alignment creation) to predict its structure.
ryhB
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 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.
