Our project mainly consists of two parts -- function module and Detection module.The role of the functional energy module is to build a low oxygen environment in the cell. The detection module functions to detect the oxygen concentration in the cell, in order to know whether the intracellular oxygen concentration has been reduced to a low level.
For the construction of a low-oxygen environment, we proposed two ideas: consuming the oxygen in the cell or transporting it to go elsewhere.
At the beginning of the experiment, we planned to transport the intracellular oxygen to build a low oxygen environment.Given this, we initially planned to use vitreoscilla hemoglobin(VHb) and transfer it into to E.coli to build the low oxygen environment in cell. However, after investigation and research, it was found that the main function of VHb is to trap oxygen and strengthen the oxygen utilization rate in the low oxygen environment. This does not fit our low oxygen construction purpose, and therefore, we abandoned the use of VHb at the beginning. We also considered human hemoglobin or myoglobin. But In the end, after we read and analysed plenty of articles, we finally choose leghemoglobin to carry and convey oxygen.
We found a special protein, leghemoglobin, from soybean rhizobia. It can directionally transport oxygen close to the cell periplasm to provide energy for cell growth. In that way, it can achieve the purpose of reducing the intracellular oxygen concentration by transporting oxygen.
But leghemoglobin comes from soybean, which means it contains introns. Therefore, we removed the introns in the process of reorganizing E. coli, allowing leghemoglobin to work normally. Later, we did codon optimization according to the codon preference of E. coli. As shown in figure 1.
Figure 1: Intron removal and codon optimization of leghemoglobin
The main role in leghemoglobin is played by heme, but heme may not be synthesized sufficiently in E. coli, so leghemoglobin may not bind to heme sufficiently after expression, and may have limited ability for oxygen delivery. For this problem, we want to solve it by adding heme to improve the transport capacity of bean hemoglobin for oxygen, and related studies have shown that this method is feasible, and the difference in expression content is shown in Figure 2. Since heme is difficult to order commercially, we used hemin for addition instead. Hemin is very similar to heme, and can be converted into heme.
Figure 2: Effect of hemin addition on leghemoglobin expression[1]
The active center of laccase CueO is copper-containing [Cu(MeCN)4]+. Initially, we did not add copper ions within the culture medium when expressing CueO. Experiments showed that it could have some effect, but it was not significant, as shown in Figure 3.
Figure 3: Fluorescence intensity comparison between control group and laccase group without copper ion
After reviewing a lot of information and talking with our teacher Yu Yang, we determined that the successful expression of laccase requires the addition of a certain amount of copper ions within the culture medium. We tried adding 0.2 mmol/L and 0.5 mmol/L of copper ions to the medium. We found that the fluorescence intensity of the group with the addition of 0.2 mmol/L copper ions was significantly enhanced when laccase was acting alone, as shown in Figure 4, which confirms our idea.
Figure 4: The fluorescence intensity of bacteria that are introduced nirB, CueO+nirB+0,0.2,0.5mmol/L Cu2+
We designed and built function modules for the experiments, i.e., laccase and soy hemoglobin were introduced into E. coli strain BL-21, respectively, along with the nirB-mRFP recombinant plasmid capable of detecting intracellular oxygen concentration. After testing for fluorescence and OD, the results are shown in Figure 5.
Figure 5: Changes of OD and fluorescence intensity with time when laccase or leghemoglobinis introduced separately
When the experiment came to this point, we thought that the effect of reducing oxygen concentration brought by the introduction of laccase and leghemoglobin failed to achieve our goal. On account of the difference between the leghemoglobin and laccase in principle, we wondered whether the effect of reducing oxygen concentration would be better when the two were introduced together. At this time, through data modeling, we simulated the fluorescence intensity of the bacteria that are only introduced leghemoglobin or laccase, which is basically consistent with the experimental data. Therefore, we simulated the fluorescence intensity of the bacteria that are introduced both leghemoglobin and laccase in the same way, which is nearly twice as high as that of the bacteria that are only introduced a single element. As is shown in the figure 6.
Figure 6: Oxygen consumption rate of bacteria with CueO (left), Lb (middle), CueO+Lb
(right).
It shows that bacteria that are introduced both Lb and CueO have the highest oxygen consumption
rate.
Therefore, we conducted cotransformation experiments under the guidance of modeling.
Both plasmids were introduced at the same time, and the experimental results showed that better results were successfully obtained, as shown in Figure 7.
It can be found that the fluorescence intensity can reach up to nearly 100,000, which is significantly higher than that of the strains introducing leghemoglobin or laccase alone, indicating that the two proteins used together play a very good role. On the other hand, the amount of bacteria shown by OD is not greatly influenced. (click here to know more about results)
Figure 7: Fluorescence and OD change with time when leghemoglobinand laccase work together
Since we could not directly detect the oxygen concentration in the cell, we intended to construct a detection module to visually show whether the low oxygen environment was constructed or not.
For the construction of a low-oxygen environment, we proposed two ideas: consuming the oxygen in the cell or transporting it to go elsewhere.
The constructed detection module needs to meet two requirements: one is resistant to low oxygen environment, and the other is able to respond to low oxygen environment. For requirement 2, we plan to attach fluorescent proteins to the requirement one to react to the low oxygen environment. After investigation, we finally chose nirB, which is sensitive to oxygen and resistant to low oxygen environment, binding with a fluorescent protein to construct our low oxygen response module.
In the course of the nirB investigation study, we found an ArcA promoter that was also resistant to low-oxygen environments. Unlike nirB, ArcA is a promoter sensitive to the rate of oxygen consumption. Although nirB has completed its corresponding function for low oxygen, the emergence of ArcA allows us to be able to react cellular oxygen consumption rates in response to low oxygen, characterizing the activity of the cells. But since the ArcA is a module coming from the icd promoter, For the icd promoter that contains not only cra modules but also the ArcA. What's more, ArcA overlap with cra. This creates the dilemma of our inability to extract ArcA alone during the PCR process. Therefore, in the actual experiment process, we chose to use the icd promoter instead of ArcA for the detection of the oxygen consumption rate. We hope to find ways to extract ArcA separately in later experiments to eliminate the interference of cra to the experiment.
That is all about the building of the detection model. After building the detection module, how to judge whether the detection module is really effective has become a difficult problem. At the beginning, we planned to use the device shown in Figure 8 to build the external low oxygen and see whether the detection module is effective. The specific working principle is as follows: Put the liquid culture medium in the bottom of the cone bottle, charge the nitrogen and other inert gas into the bottle through the long gun head, and blow out the oxygen in the culture medium to ensure the low oxygen in the cell growth environment; then, removing the longer gun head (as shown in Figure 9), the inert gas such as nitrogen is passed through the shorter gun head to ensure the overall low oxygen of the whole tapered bottle.
Figure 8: low oxygen device 1
Figure 9: low oxygen device 2
However, because it is more difficult to access nitrogen with the above method in the school laboratory. So we improved the experimental setup (Figure 10, Figure 11) -- After vacuuming the anaerobic bag, Add a protective gas generation agent to it, and seal the protective gas to create a low oxygen environment.
Figure 10
Figure 11
Connect the nirB and icd ArcA modules in series to a plasmid vector (the circuit is shown in Figure 12). This can reduce the number of plasmids, and also reflects the characteristics of synthetic biology that can integrate different modules. According to the principle of chemical reaction, the higher the oxygen concentration, the higher the oxygen consumption rate. As stated in the experimental design, the lower the oxygen concentration, the higher the nirB activity; The lower the oxygen consumption rate, the higher the ArcA activity. Therefore, the ideal state is that when the oxygen concentration and oxygen consumption rate are high, the red and green fluorescence intensities are weak; when the oxygen concentration and oxygen consumption rate are low, the two fluorescence intensities are strong. The fluorescence intensity of the two bands measured by the microplate reader can reflect the oxygen concentration and oxygen consumption rate, which can improve the detection efficiency.
Figure 12: Plasmid vector constructed in tandem by nirB and ArcA
According to some teacher's advice, We plan to use laccase to reduce the oxygen concentration first, and then then further reduce the concentration of oxygen with leghemoglobin. The reason is that laccase is an enzyme, which can play a role in high oxygen concentration and consume a lot of oxygen; However, leghemoglobin is combined with oxygen. If the oxygen concentration is too high, it will be saturated with leghemoglobin, so it can not have a good oxygen reduction effect. Therefore, laccase is suitable for decreasing oxygen at high oxygen concentration, and leghemoglobin is suitable for decreasing oxygen at low oxygen concentration.
Figure 13: Schematic Diagram of rocking equipment
Figure 14: The final effect of the rocking equipment
Therefore, inspired by the use of gene oscillator by BUCT China team, we designed a metabolic pathway based on the principles of synthetic biology (Fig. 13). Among them, O2 sensitive promoter I/II (hereinafter referred to as OS promoter) is a hypoxia inducible promoter. The oxygen concentration of OS promoter I is higher than that of OS promoter II. It is assumed that the oxygen partial pressure required for induction is p1 and p2 respectively. T7 promoter guides the expression of laccase, OS promoter I promoter guides the expression of leghemoglobin, and OS promoter II is used as an anti-sense promoter[2], which is reversely connected to laccase and leghemoglobin.
At first, laccase and leghemoglobin were not expressed. Then we used IPTG induction to make laccase begin to express and reduce intracellular oxygen concentration. When the partial pressure of oxygen decreases below p1, OS promoter I is activated and leghemoglobin is expressed. When the partial pressure of oxygen further decreases below p2, the oxygen concentration no longer needs to be further reduced. Therefore, OS promoter II is also activated and starts to reverse express, producing anti-sense mRNA chains that complement and pair with the original mRNA chains, so that the translation process of leghemoglobin and laccase is inhibited, thus realizing regulation. After a period of time, the oxygen concentration gradually recovers, and the activity of OS promoter II decreases. Transcription and translation begin to work normally, and the oxygen concentration starts to decrease again, so as to achieve the effect that a gene expression oscillates with time.
In the period of high oxygen concentration, cells mainly grow and reproduce. In the period of hypoxia, the cells mainly produce hydrogen and nitrogen fixation. Thus, the cycle of growth and production is realized, as is shown in figure 14.
Since the use of VHb to transport oxygen to reduce the intracellular oxygen concentration did not work, we turned our eyes to the direction of consuming the intracellular oxygen. After the investigation and study, we finally intend to choose laccase to build a low oxygen environment. Laccase is a reductive enzyme that can catalyze the reaction of oxygen with reducing substances to achieve the effect of consuming intracellular oxygen. Since we finally intended to conduct experiments in E. coli, the laccase we selected was the polycopper oxidase -- CueO in E. coli. Based on the above ideas, we started the experiment.
Later, we found that the laccase would be localized to the cellular periplasm. The main reason is that the laccase contains a 28-residue N-terminal signal peptides.For this problem, we reconstructed the laccase plasmid to remove the signal peptide sequences contained in the laccase. The reconstructed laccase plasmids are shown in Figure 15.
Figure 15: Laccase without a signal peptide
Figures 16 and 17 show the final laccase and leghemoglobin plasmid constructs. But during the experiment, we found that the promoters used were not very ideal. Initially, we used the default hypoxia promoter, but the coupling is complex.For control convenience, we redesigned both of the promoter araBAD and tacThe araBAD promoter is a promoter from Escheric. Coli, transduced growth to 0.6-0.8OD, the inducer was arabinose at an induction concentration of 7.9%. The tac promoter is a promoter heterozygous for the trp and lac promoters, transduced growth to 0.6-0.8OD, the induction was IPTG at an induction concentration of 0.1%. These two promoters are universal, and the mechanistic studies are relatively clear. However, due to insufficient experimental time, we finally did not test the induced expression effect of these two primes in the actual experiment. We hope to be able to refine this part in the subsequent experiments.
Figure 16: laccase plasmid
Figure 17: leghemoglobin plasmid
In subsequent experiments, we hope to allow for a detailed partition of the promoter function. When the intracellular oxygen concentration is high, hyperoxic promoter can be used to induce the expression of bean hemoglobin alone, making the laccase not working. However, when the intracellular oxygen is low, low oxygen promoters such as nirB are able to induce laccase expression alone, making leghemoglobin not work。Finally, making the leghemoglobin and laccase play a division of labor and cooperation effect.
This project has confirmed that the introduction of leghemoglobin can reduce the intracellular oxygen concentration, but there is still doubt whether leghemoglobin can deliver oxygen to the respiratory site. Therefore, in the subsequent experiment, it is necessary to investigate whether the leghemoglobin has the function of transporting oxygen to the respiratory site. If not, it is necessary to introduce a signal peptide to locate the leghemoglobin on the cell membrane in the later stage, so as to deliver oxygen to the respiratory site.
We "oxygen hunter" have added leghemoglobin (that transports oxygen) and laccase (that consumes oxygen) into cells, but if we add some parts that enrich oxygen (eg. VHb) (figure 18) into cells,we can also fulfill the intracellular high oxygen pressure environment. By using different promoters, the devices that regulate oxygen can work together, so that the oxygen concentration in cells can be controlled in different ranges. Finally we can construct an oxygen controller system.
We hope our project can achieve our second idea and achieve the effect of "oxygen controller".
Figure 18: the molecular structure of VHb
[1]Yue Su. Study on efficient expression of heterologous leghemoglobin in microorganisms(2020)
[2]Grant Logan , Christine M. Smyth, et al. 298. Characterization of a Novel Anti-Sense Promoter Element in Adeno-Associated Virus: Implications for Hepatocellular Carcinoma(2016)Molecular Therapy