Measurement

We use fluorescent protein to connect promoters and artificially create a hypoxic environment to detect whether the detection module can work.

In the experiment of the detection module, we first used chloramphenicol to screen the engineering bacteria that successfully imported the required original components, that is, the bacteria containing chloramphenicol resistance.

Under the condition of continuously inflating into the bottle, although the oxygen concentration can be kept as low as possible, the mouth of the bottle is not sealed. Being exposed to the open air, although there is an outward positive pressure to make the air flow outward, it cannot be ensured that no foreign bacteria will enter the bottle through the open mouth, polluting the liquid culture medium, or even killing the engineering bacteria therein; At the same time, in order to achieve a long-time anaerobic state, the gas needs to be continuously flushed, which is a challenge to the requirement of gas quantity. And also need someone to keep the side, not only waste inert gas, but also waste a lot of time, therefore, after the teacher's opinion and correction, we decided to abandon this plan.

And for the vacuum, by the protective gas generator released the appropriate amount of inert gas plan, because the operation can not balance the vacuum and pump does not touch, pollute the experimental object, to avoid destroying the culture conditions of bacteria on the culture dish, was put aside. Meanwhile, for the lack of large isolation boxes, we could only perform small-scale experiments. The existing protective gas generators on the market are similar to NaH that produces hydrogen, NH4NO3 that produces N2 or so produce too much or too little gas, which is not suitable for the smaller experiments we need to do.

At the same time, if you want to adjust the concentration of oxygen, you need oxygen agents such as CaO2. However, due to our lack of understanding of the above chemicals, it is impossible to clearly determine the number and concentration of chemicals required for the corresponding concentration of the gas. Under the guidance of our teachers, we decided to use the existing culture medium preservation bags, anaerobic generators, micro aerobic generators, and carbon dioxide gas generating bags produced and sold by the company to build our hypoxic environment. The general control of oxygen concentration is realized through deep orifice plate shakers with different rotating speeds.

In the anaerobic experiment, due to the small volume of the anaerobic bag and the inability to ensure that the surface of the test bottle remains sterile, we decided to conduct only the hypoxia experiment of the solid culture dish according to the instructions of the anaerobic bag to prove that the nirB promoter and red fluorescent protein can be normally expressed under hypoxia. Due to the existence of anaerobic bags, the amount of fluorescent protein in the two media should be roughly the same, but the hypoxia media is relatively higher and brighter. Our experimental results also prove this. We found that colonies present on plates placed within the anaerobic bag, showed red fluorescence under the UV light. This is a clear proof that the red fluorescent protein is indeed expressed, and the nirB promoter can drive it.

In the experiment of time-sharing sampling and measurement, we used normal LB culture medium and the hypoxia culture medium we configured to conduct the experiment. The normal culture medium was used as the control group, and it was continuously cultivated at 37 ℃. At the same time, according to the shaking speed of the shaking table, we divided the experiment into several different groups. Each group took a sample every eight hours after the bacteria grew, a total of three samples, that is, 24 hours. The data of fluorescence intensity under different time gradients can infer the expression trend of fluorescent protein with oxygen concentration.

According to our prediction, as the shaking rate and the culture time increase, the dissolved oxygen concentration in the liquid LB medium should be in a rising state, possibly reaching saturation after a certain time. However, because the expression of promoter increases with the decrease of oxygen concentration, and the expression amount decreases with the increase of oxygen concentration, the expression amount in unit time will gradually decrease, so the total expression amount will form a rise, but the rising speed will continue to slow down, and tend to a stable image. As for the presence of cysteine-containing medium, the Cys can absorb a fraction of oxygen. At the beginning part, the dissolved oxygen concentration always maintains a low state, while the expression of the fluorescent protein is also always more abundant, forming a straight line with a high slope. After cysteine depletion, the experimental status became similar to LB medium. And our experiments just confirm this point.

Escherichia coli in LB medium has a high oxygen concentration and generates less carbon dioxide when it carries out aerobic respiration; In the LB Cys medium, the oxygen concentration is low, and anaerobic fermentation produces more carbon dioxide, forming bubbles.

Because we are not familiar with the Clark electrode method to measure the intracellular oxygen consumption rate, and the laboratory has no relevant equipment for us to measure, we choose to measure the cell oxygen consumption rate by measuring the ArcA activity affected by the icd promoter.

The successful expression of the detection module can be used to reflect the effect of the function module.

In the experiments with the function modules, we used SDS-PAGE to examine the expression of bean haemoglobin and laccase.

Protein electrophoresis instrument

We introduced laccase or leghemoglobin into Escherichia coli BL-21 strain together with the plasmid containing nirB fluorescent protein to reflect the construction effect of hypoxic environment with fluorescence intensity.

Experimental results diagram

We used fluorescence microscopy to observe the fluorescence intensity, which can reflect the location of the relevant protein distribution, as well as the intracellular oxygen concentration distribution. Through this, we can verify the concept of our project.

Because the data measured in our experiment is fluorescence intensity, we need to calculate the oxygen consumption rate of leghemoglobin by fluorescence intensity and dissolved oxygen concentration. For this purpose, we deduced the mathematical relationship between the fluorescence intensity and the oxygen consumption rate of leghemoglobin. We put the derivation process in the Model part of our website.