In the detection module, we first used the oxygen sensitive promoter nirB. Its characteristic is that the lower the oxygen concentration, the higher the expression activity. It is a gene sequence with more than 2000 bp, and its activity under hypoxia is much higher than that under normal oxygen concentration. nirB's principle operation is there are two critical regions on nirB for induction by anaerobic conditions. The first is a hexamer region, TAAGGT, which is necessary for anaerobic activation. The second region is the fumarate and nitrate reductase regulatory (FNR) protein-binding site. FNR protein is a general regulatory protein that activates transcription initiation in some anaerobic promoters such as nirB under anaerobic conditions.
We then inserted the red fluorescent protein (mRFP) gene into the plasmid vector p5T7 and transferred it to E. coli. In this way, by detecting the fluorescence intensity in the red light wavelength range, the intracellular oxygen concentration can be reflected, thus realizing a convenient and high flux detection. That is, the stronger the fluorescence, the higher the nirB activity, the lower the oxygen concentration.
nirB-red fluorescent protein expression vectors
We use anaerobic bags with different oxygen concentrations, or use hypoxic culture media. First, we detect the corresponding fluorescence intensity under different oxygen concentrations by conducting gradient hypoxia tests, and then build a detection system. We can reasonably extrapolate the existing data. Later, we can predict the oxygen concentration in the system at that time by directly measuring the fluorescent protein activity, After that, we will judge and detect the hypoxia effect of the system we built, and visualize the data that is difficult to be visualized, so as to vividly display our experimental results.
The second part of the detection module uses icd promoter and ArcA transcription factor to detect oxygen consumption rate. The experiment showed that the activity of ArcA had no significant relationship with oxygen concentration, but had a significant negative correlation with oxygen consumption rate (R2=0.93)[1]. This is because ArcA is closely related to cell respiration and oxygen metabolism.
ArcA activity is regulated by fermentation products via the inhibition of ArcB phosphorylation. Since the NADH/NAD+ ratio increases under oxygen limited conditions, fermentation reactions occur to facilitate NADH regeneration. The accumulated fermentation product(s) would inhibit ArcB phosphorylation. The measurement of the phosphorylated state of ArcA and ArcB would make this mechanism more reliable.
Relationships between ArcA activity and qO2.[5]
The structure of icd promoter
We extracted the icd promoter and the ArcA fragment after it, and connected it with green fluorescent protein (eGFP) to measure its expression activity, then transferred it to plasmid p5T7, and then transferred it to E. coli. Find the method to calibrate the oxygen consumption rate. In this way, the lower the oxygen consumption rate, the higher the fluorescence intensity.
icd-green fluorescent protein expression vectors
Similarly, we artificially constructed a culture environment with different oxygen concentrations to detect the functions of icd and ArcA modules. However, due to time constraints, we have not been able to further explore the relationship between ArcA activity and oxygen consumption rate. As expected, we will rely on different oxygen concentrations and their corresponding oxygen consumption rates to obtain a chart that can accurately represent the degree of oxygen concentration decline and oxygen consumption efficiency by curve fitting through software, so as to judge the effectiveness of the system we have built.
In order to detect the effects of nirB and icd, we need to create a hypoxic environment artificially. After our engineering improvement, we purchased anaerobic bags and configured anaerobic culture media with a specific formula. Among them, the anaerobic bag uses specific substances to absorb oxygen, thus creating a hypoxic environment in a closed environment inside the bag, while the anaerobic culture medium contains a large amount of reducing substances.
anaerobic bag we used
After these specific promoter sequences are inserted into specific fluorescent proteins, they are introduced into the plasmid p5T7, and then inoculated onto the solid medium with a lattice. They are cultured under normal conditions and in anaerobic bags that create an oxygen poor environment, respectively. After a period of time, take out the medium in the anaerobic bag, and pick it together with the normally cultured Escherichia coli in the super clean bench to a new culture dish for the convenience of subsequent fluorescence detection. Pick up some strains on the cover glass, place the cover glass in the dark room under a fluorescent microscope with a magnification of 400X and observe through the computer screen.First, find the places where the strains are enriched in large quantities in the normal light band. Then turn on the excitation light source in the blue light band, and observe it in the red light band and green light band. It can be found that the nirB promoter emits red fluorescence, and the icd promoter emits green fluorescence. At the same time, The fluorescence intensity of bacteria cultured in anaerobic bags was significantly higher than that of bacteria cultured in normal atmosphere with 21% oxygen concentration.This can prove that the nirB and icd promoters can indeed express, and can express better under hypoxia.
After that, we prepared normal and hypoxic liquid culture media, respectively, and connected the nirB and icd promoters into them, placed them in a shaker for culture, and took samples at regular intervals. Finally, put it into the microplate reader for detection, and measure its absorbance and fluorescence intensity under different wave bands. The specific effects of hypoxia on the two promoters can be obtained more accurately.
Intracellular oxygen concentration and oxygen consumption rate can be used to characterize the status of intracellular oxygen metabolism, provide data support for modeling work, and can later cooperate with functional module.
In the function module, we used legemoglobin and laccase to reduce the oxygen concentration in cells.
leghemoglobin, which exists in soybean root nodules in nature, is a protein composed of 144 amino acids. rhizobia need oxygen to supply energy, but nitrogenase activity is easily inhibited by oxygen. The leghemoglobin in the root nodule can transport oxygen to the cell respiration site in a directional manner, provide sufficient energy for nitrogen fixation, and reduce the oxygen concentration around the nitrogenase to ensure the nitrogenase activity[2].
We introduced the leghemoglobin gene into E. coli cells for recombinant expression. We did codon optimization for leghemoglobin gene, and integrated it into the plasmid pET-28A (+) with lactose operon. We introduced the recombinant plasmid into E. coli BL-21 strain for culture.
Leghemoglobin expression vectors
Laccase is a kind of oxidoreductase which widely exists in microorganisms, animals and plants. Its basic principle is to catalyze the reaction of reductive substrate and oxygen to reduce the concentration of oxygen, which can directly consume oxygen, thus helping to build a hypoxic environment in cells[3]. It is a 516 amino acid protein, of which the first 28 amino acid residues are signal peptides, which can locate CueO in the periplasm[4].
We are going to choose the multiple oxidase CueO contained in Escherichia coli as laccase. We constructed the recombinant plasmid pET-28A(+)-CueO and transferred it into E. coli BL 21(DE3) strain for culture.
Ribbon representation of the CueO molecule (PDB code 1N68). (a) Overall structure highlighting three structural domains (D1, red; D2, green;D3, gold), four defined copper centers (T1, blue; T2, teal; T3 (binuclear), purple; T4, red), and the location of methionine residues (in gray-yellow sticks).(b) Molecular structure around T4 highlighting the ligands for CuII, possible ligands for CuI (including M440), and a hydrogen bond connecting the T4 copper ligand D439 and the T1 copper ligand H443[4].
We have observed the experimental effect by combining the function module and the detection module during the experiment. The basic principle is that when induced with IPTG, CueO or leghemoglobin will be expressed in large amounts, thus reducing the intracellular oxygen concentration. The detection module will detect the decrease in oxygen concentration and "report" it in the form of fluorescence production.
We constructed the plasmid of leghemoglobin and laccase, and then introduced it into E. coli and the detection module. The experimental results are detailed in the result section.
In order to promote the better expression of function modules, we also considered some problems: first, part of the leghemoglobin is heme, and we considered the influence of heme on the experiment.
We have conducted the following experimental design: we will only import one group of nirB, leghemoglobin+nirB+hemin, and leghemoglobin+nirB, and carry out a control experiment. The results are listed in the results.
Secondly, laccase contains copper ions. Adding copper ions in the culture will help laccase expression and function, and the concentration of copper ions may have a certain impact.
We have conducted the following experimental design: we will only introduce nirB, laccase+nirB+copper ions, with the concentration of 0mmol/L, 0.2mmol/L and 0.5mmol/L respectively, and carry out the control experiment. The results are listed in the results.
In the design experiment, we take into account that laccase and leghemoglobin use different mechanisms to create a hypoxic environment, which can complement each other: leghemoglobin is on the membrane, laccase is in the cell, and external input is prevented to ensure respiration; Internal leakage prevention and oxygen free. It can better solve the oxygen paradox problem.
Therefore, we introduced laccase and leghemoglobin into the chassis strain for experimental measurement, and compared with the previous experimental results. We introduced laccase+leghemoglobin+nirB, added copper ions (0mmol/L, 0.2mmol/L, 0.5mmol/L) and hemin, and carried out three groups of control experiments. See results for details.
We cultured these strains and detected the expression of fluorescent protein with fluorescence intensity to judge the actual effect of adding function modules (leghemoglobin, laccase or both) on the intracellular oxygen concentration. We judge the concentration of bacteria in the liquid culture medium by the data of bacterial liquid absorbance (i.e. OD value) to reflect the growth of bacteria. In order to reduce the chance of the experiment, we set up three parallel experiments for each group of data, and finally took the average value of the three groups as the experimental data of the sampling time of this group.
We conducted data analysis on the experimental results, and the experimental results were basically the same and basically consistent with the data after we modeled and predicted through Matlab. The growth curve of Escherichia coli grew in accordance with the S-shape. At first, the growth was slow due to the lack of strain concentration. Later, with the increase of strain concentration, it gradually reached the maximum propagation rate. Later, due to the lack of nutrients in the culture medium, intensified intraspecific competition and other reasons, the growth of the strain gradually slowed down. Through data analysis, we basically determined that laccase and leghemoglobin did indeed play a role in reducing the oxygen concentration, and successfully achieved the goal of constructing the hypoxic environment in cells, while making the growth and reproduction rate of bacteria almost unaffected. Then the effects of copper ion concentration and heme addition on the construction of hypoxic environment were analyzed.(Click here to know about result)
[1] Prost JF, Nègre D, Oudot C, Murakami K, Ishihama A, Cozzone AJ, et al. Cra-dependent transcriptional activation of the icd gene of Escherichia coli. Journal of Bacteriology. 1999.
[2] Ryu M-H, Zhang J, Toth T, Khokhani D, Geddes BA, Mus F, et al. Control of nitrogen fixation in bacteria that associate with cereals. Nature Microbiology. 2019.
[3] Djoko KY, Chong LX, Wedd AG, Xiao Z. Reaction Mechanisms of the Multicopper Oxidase CueO from Escherichia coli Support Its Functional Role as a Cuprous Oxidase. Journal of the American Chemical Society. 2010.
[4] Karrera Y. Djoko,# Lee Xin Chong, Anthony G. Wedd, and Zhiguang Xiao. Reaction Mechanisms of the Multicopper Oxidase CueO from Escherichia coli Support Its Functional Role as a Cuprous Oxidase. JACS. 2009.
[5] Reza, N., & Akbari Eidgahi, M. R. (2014). Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli. Jundishapur Journal of Microbiology, 7(6). doi:10.5812/jjm.15942