Results

First of all, we tested that the selected hypoxic starting parts had successfully played the function of responding to oxygen in E. coli.

Secondly, through SDS-PAGE, we can see that the leghemoglobin of the source plant is successfully expressed in E. coli, indicating that our codon optimization has achieved the effect; Laccase was also successfully expressed, although its molecular weight was high and its expression was low.

Figure 1: SDS-PAGE

1,2: CueO+nirB, sediment(1) and supernatant(2).
3,4: CueO+nirB+0.2mM Cu2+, sediment(3) and supernatant(4).
5,6: CueO+Lb+nirB+hemin, sediment(5) and supernatant(6).
7,8: CueO+Lb+nirB+0.2mMCu2+ +hemin, sediment(7) and supernatant(8).
9,10: CueO+Lb+nirB+0.5mMCu2+ +hemin, sediment(9) and supernatant(10).
11,12: only nirB, sediment(11) and supernatant(12).
13,14: Lb+nirB+hemin, sediment(13) and supernatant(14).

Next, we verified the function of the gene line designed by the project. From the growth curve, the strain density of the experimental group with leghemoglobin is 82.3% of the control group containing only nirB, and the number of cells with laccase is 88.6%. No matter which experimental group, compared with the control group containing only nirB,the growth rate descent of the experimental group is only about 10% to 20%, which is acceptable, because the expression of foreign protein will have a certain impact on the growth of E. coli, but there is no significant reduction.

From the perspective of fluorescence protein intensity, the fluorescence intensity of Escherichia coli introduced with leghemoglobin and laccase part was significantly higher than that of the control group, and the effect of co-introduction was more significant than that of single introduction.

Based on the functional verification of the hypoxia response part, it is proved that the two parts of leghemoglobin and laccase play a role in reducing the intracellular oxygen without affecting the cell growth, and the combination of the two parts is more effective in reducing oxygen.

First, we verified the function of the selected oxygen response parts nirB and icd.

In the functional verification of nirB part, we created a hypoxic environment through the cysteine culture medium. As an experimental strain, the control strain was cultured under normal oxygen concentration. The OD600 of the two groups of strains were measured at different culture times, and the red fluorescence intensity of the experimental group and the control group was detected after 22 hours of culture.

From the experimental results, after introducing nirB part into E. coli, compared with the control strain without part, the OD value has no significant change in its growth process, which proves that nirB part has no effect on the growth of E. coli. When cultured for 22 h, the test results of fluorescence microscope showed that the strains in the experimental group after the introduction of nirB part did produce red fluorescence. Moreover, through the detection of fluorescence intensity, the red fluorescence intensity of the experimental group was significantly higher than that of the control group, which proved that the hypoxia response part nirB we selected successfully played its role, and the ability to express genes under hypoxic conditions was higher than the normal oxygen concentration.

In the function verification of icd part, we mainly verified the reaction of oxygen consumption rate of the part. In the hypoxic culture environment created by using cysteine medium, the change of fluorescence intensity with cell growth after the introduction of icd part was detected. From the experimental results, it can be seen that the cells enter a stable phase after 12 hours, and the cell growth before the stable phase is a logarithmic phase. At this stage, the cells grow rapidly and the oxygen consumption rate is high. It can be seen from the intensity of green fluorescent protein that the intensity of green fluorescent protein decreases with the logarithmic growth process, which proves the negative correlation between icd and oxygen consumption rate, and verifies that the icd part has successfully played its role. At the same time, it can be seen from the results of fluorescence microscopy that the green fluorescence in cells was indeed expressed in the late logarithmic growth period of cells under the condition of hypoxia culture medium.

Figure 4: OD and fluorescence intensity of icd-Cys

Figure 5: icd Observation image by fluorescence microscope

The OD value of the experimental data was measured from the third hour to the end of the 12th hour, that is, six measurements were made, while the fluorescence intensity was measured from the fourth hour to the end of the 12th hour, a total of five measurements were made.

In the experimental group of leghemoglobin (as shown in Fig. 6), the addition of hemin can significantly reduce the intracellular oxygen, which is 29.6% lower than that of the control group only introducing nirB. After adding hemin in the culture, we found that the growth curve of the experimental group with hemin was higher than that of the experimental group without hemin, indicating that the direction of adding hemin in our engineering iteration was correct.

Figure 6: Fluorescence intensity and OD of bacteria with Lb,with or without hemin

In the laccase experimental group (as shown in Figures 7), we first conducted the experiment of only introducing laccase. The oxygen reduction efficiency was only 9.9%, and the growth was also affected. Later, through literature review, it was found that adding copper ions would help laccase catalysis, so we carried out the experiment of adding 0.2 mmol/L and 0.5 mmol/L copper ions. It was found that the oxygen reduction efficiency of the experimental group added 0.2 mmol/L copper ion reached 37.3%, significantly improving the laccase effect; Although the experimental group added 0.5mmol/L copper ion is better than 0.2mmol/L in growth, it is obviously insufficient in oxygen reduction efficiency.

Figure 7: Fluorescence intensity and OD of bacteria with CueO, adding 0/0.2/0.5 mmol/L Cu²⁺ in the culture

Because leghemoglobin and laccase are different in the principle of consuming oxygen, if co culture complements each other in effect, the effect of reducing oxygen will be better than that of introducing them alone, so we carried out combined expression. In the experimental group containing both leghemoglobin, heme and laccase (in Figures 8), the concentration of 0.5mmol/L copper ion has the largest role in promoting the production of hypoxic environment, while the effects of different concentrations of copper ion on hypoxic environment are different. Considering the effects of cell growth and oxygen reduction, we chose the experimental group of 0.5mmol/L copper ion. Its oxygen reducing effect reached 45.8%, which was 19.2% and 8.5% higher than that of single leghemoglobin and laccase, respectively. It proved that the combined expression effect was better than that of single functional part.

Figure 8: Fluorescence intensity and OD of bacteria with Lb and CueO, adding hemin and 0/0.2/0.5 mmol/L Cu²⁺ in the culture

At the same time, we found an interesting phenomenon in the experiment. When 0.5mmol/L copper ion alone acts with laccase, the oxygen reduction effect is not good, but after the introduction of leghemoglobin, the best effect is achieved. Our guess is that copper ions can promote the expression of leghemoglobin to a certain extent. Although 0.5 mmol/L copper ions have poor oxygen reducing effect, they may promote the expression of leghemoglobin to a greater extent, making it a group that plays the largest role in promoting the expression of leghemoglobin when both leghemoglobin and laccase are contained. Later, we also found a similar phenomenon in a literature, which supports our guess.

In our communication with teachers, the above four analyses were praised and affirmed by Dr. Yang Yu.