Team:XHD-Wuhan-A-China/Human Practices
Human Practices
How can genetic engineering solve real life problems, which was the core theme of the iGEM competition and the answer we were all looking for together, through background checks, expert interviews, experimental design and field trials, we finally created this project within our power.
Background research
Soil is the substrate on which human beings live and the foundation on which all things grow. However, due to the process of industrialization in the human world, the natural environment inevitably changes, especially the soil environment. Due to large-scale artificial irrational cultivation and the abuse of chemical fertilizer, soil compaction, soil structure change, porosity decrease, hardness increase and infiltration capacity decrease, resulting in the degradation of soil fertility characteristics. Fertilizer abuse leads to soil salinization and acidification, which is one of the important causes of soil compaction. We hope that through our engineering bacteria can solve the soil problems caused by soil acidification and salinization, to protect the soil environment and farmland security to provide technical guarantee.
Interview Professor. Ren for project design
After we determining the direction of our object, we are wondering that how to use the knowledge to solve the problem in an efficient way. Therefore, we interviewed a biology expert who is specialized in microorganism. He said that sources of nitrate in soil include chemical fertilizers, plant humus, rain (a small amount of nitrogen is reduced due to lightning), and biological nitrogen fixation. One major source is fertilizer, which is the most easily controlled factor in the experiment when we mentioned our project and our opinions to him. He suggested only considering fertilizers during our experiments, so we tried to do like he recommended.
Moreover, we need to add the appropriate nitrate concentration to the culture medium, but we are not quite sure how the concentration should be determined. So we asked the experts for help. He said that 0.5-1mm KNO3 is added to the nitrogen-free medium, with a maximum of 2 mM generally.
Questionnaire survey
Our professors and experts agree that our project make sense and the future of our project is available. But we still need to improve our project, so we designed a questionnaire to understand the attitude of public to our project about reduce the salt content in the soil by decomposing nitrate in the soil into a gaseous state.
The results of some of the questions are follows:
Question 7: Are you interested in our program?
Question 9: Do you think it is possible to reduce the salt content in the soil by decomposing nitrate in the soil into a gaseous state?
Question 10: Whether our project make sense?
Question 11: 11.Do you know anything about iGEM?
Selection of biological chassis
In iGEM projects in previous years, Escherichia coli was usually used as a biological chassis, and fewer rhizobia were used as a biological chassis, mainly because of their slow growth rate and inefficient conversion. As a participant in the natural nitrogen cycle, rhizobia essentially has the dual functions of nitrogen fixation and denitrification and is a natural biological chassis involving nitrogen cycle circuit projects. We have initially designed a gene expression system suitable for rhizobia, which provides a reference for relevant research.
Interview Professor Ren
Through preliminary investigation, we found that the concentration of nitrate in soil will have a certain impact on the nitrogen fixation of rhizobia. So what is the impact of denitrification on biological nitrogen fixation and natural nitrogen cycles? To understand this problem, we had the honour to interview Professor Ren.
We learned that nitrate concentrations in the soil will affect nitrogen fixation by rhizobia because the soil is high in nitrate-nitrogen, which is easily met by crop roots but still requires nitrogen fixation by rhizobia. As rhizobia and plant roots are in a symbiotic relationship, with rhizobia fixing nitrogen for plant consumption and the carbohydrates formed by plant photosynthesis nourishing the rhizobia, each is beneficial to each. The roots can now readily take up nitrogen on their own, and there is certainly less dependence on fixed nitrogen. Denitrification is another concept. Under anaerobic conditions, nitrifying microorganisms do not work well. At this time, denitrifying microorganisms come out to do their work. Nitrogen oxides are also greenhouse gases. Their warming effect on the atmosphere is 200-300 times that of carbon dioxide.
Complete interview Q&A:
Q1:We need to add the appropriate nitrate concentration to the culture medium, but we are not quite sure how the concentration should be determined.
A1:Generally, 0.5-1mm KNO3 is added to the nitrogen-free medium, with a maximum of 2mM.
Q2:We also want to know what is the main source of nitrate in the soil, so if we know the source, we can better design experiments.
A1:Sources of nitrate in soil include chemical fertilizers, plant humus, rain (a small amount of nitrogen is reduced due to lightning), and biological nitrogen fixation. One major source is fertiliser, which is the most easily controlled factor in the experiment. I recommend only considering fertilizers.
A2:Nitrate in soil mainly comes from nitrogen fertilizer applied to soil, common nitrogen fertilizer includes urea, ammonium sulfate, ammonium chloride, ammonium dihydrogen phosphate and compound fertilizer. Under the action of nitrifying microorganisms, nitrogen fertilizer was transformed from ammonium nitrogen to nitrate nitrogen. If urea is applied, the urea is first hydrolyzed to ammonium nitrogen by urease, which is then oxidized to nitrate nitrogen. The nitrification process mainly occurs in the dryland soil with good aeration conditions. If it is paddy soil, due to the anaerobic environment, the nitrification process is weak at this time, so the nitrate (nitrate nitrogen) in paddy soil is very low, while the nitrate nitrogen in dryland soil is relatively high.
Q3:We need to investigate the current arable land damage caused by soil compaction, the economic loss and the impact on farmers, and want to know how to obtain relevant information and specific data.
A1:Strictly speaking, soil compaction is caused more by farming than by fertilizer itself. Fertilizers also play a role, such as ammonium sulfate, which is a physiologically acidic fertilizer. Crops take up the preferred ammonium nitrogen, leaving sulfate roots in the soil. When sulphate radical accumulates in soil, it can also destroy soil structure. There are several key parameters of soil consolidation that you can refer to, one is that the bulk density of the soil will increase, the second is that the soil capillary space will decrease, and the third is that the soil aggregate structure will get worse. Specific standards and parameters also need to consult the literature.
A2:Compaction problem is one of the current soil degradation problems, mainly is for a long time not science, excessive use of chemical fertilizer led to the decrease of the soil organic matter and calcium plasma leaching, eventually led to the deterioration of soil structure and harden, the microbial technology can improve soil harden and but the premise is to increase soil organic matter, because of microbes to organic matter for food; There are many studies on soil compaction on cultivated land destruction, but there may be little information on the impact of soil compaction on farmers' economic loss, which can be collected through literature review.
Q4:Through the preliminary investigation, we found that the concentration of nitrate in the soil would have a certain impact on the nitrogen fixation of rhizobia, so what is the impact of denitrification on biological nitrogen fixation and the natural nitrogen cycle?
A1:Nitrate concentration in the soil will affect the nitrogen fixation of rhizobia, because there is a lot of nitrate nitrogen in the soil, crop roots can easily meet, why also need rhizobia nitrogen fixation? Because rhizobia and plant roots are in a symbiotic relationship, rhizobia fix nitrogen for the plant to eat, and carbohydrates formed by plant photosynthesis nourish rhizobia, everyone benefits. The roots can now easily absorb nitrogen on their own, and of course are less dependent on rhizobia for nitrogen fixation. Denitrification is another concept. In anaerobic conditions, the nitrifying microbes can't do their job well. At this point, the denitrifying microbes come out and do their work. Nitrogen oxides are also greenhouse gases, and their warming effect on the atmosphere is 200-300 times greater than that of CO2. The effect of denitrification on the natural nitrogen cycle has always been a hot research topic in soil and environment.
A2:Your research is correct. When there is too much nitrogen fertilizer in the external environment, the symbiosis efficiency of legumes and rhizobia is reduced, and the invasion of rhizobia is reduced. Even if the nodule that can fix nitrogen has been formed, the efficiency of fixing nitrogen of these nodule is greatly reduced under the external inorganic nitrogen fertilizer condition, and the senescence of nodule is greatly accelerated.
Bacterial denitrification can indeed convert nitrate into nitrogen, but the effects of denitrification on biological nitrogen fixation have been little studied. Denitrification plays an important role in the natural nitrogen cycle. Excessive nitrate brings great harm and pollution to soil. Denitrification can oxidize excessive nitrate compounds (fertilizer, biological nitrogen fixation) into nitrogen, which is beneficial to the nitrogen cycle in nature.
Integrated Human practices
Our project was inspired by the significant environmental change by development, from the incipient concepting to the final stage of our project, human practice activities have played an important role.
1. Many scientists believe that solving soil problems by soil acidification and salinization is a primary step for continuous human development. Besides the development of synthetic biology is crucial on improving soil quality.
2.After analyzing the results from the online questionnaire survey,we found out that over 75% of people,students from high schools and universities,interested in our project and thought it's meaningful.
3.During the interview with professor Tian,a biology expert ,he told us that sources of nitrate in soil include chemical fertilizers, plant humus, rain (a small amount of nitrogen is reduced due to lightning), and biological nitrogen fixation. One major source is fertiliser, which is the most easily controlled factor in the experiment. I recommend only considering fertilizers.
4.After learning iGEM soil improvement project, We chose Sinorhizobium fredii HH103 acts as the base organism. NapA and nirK genes are present Genes in rhizobia that are closely related to rhizobia denitrification. This project will include napA or nirK through synthetic biology techniques. The gene expression plasmid is introduced into the rhizobia, hopefully in this way root strengthening by increasing the copy number of napA or nirK genes in rhizobia bacteria denitrification, thus better decomposition of excess soil nitrate alleviates the hardening effect of soil salinization. The genetic background of Sinorhizobium fredii HH103 is not as good as that of Escherichia coli.
5.However the experiment was not rising all the way. The research of Sinorhizobium fredii is not clear, so you may encounter various unexpected problems. And Sinorhizobium fredii the growth cycle of HH103 is longer than that of Escherichia coli, culture time and experiment the process will be more time-consuming. In this project, we have completed most of experiments. But, does the denitrification enhancement type Sinorhizobium fredii HH103 can survive in the natural environment? The effect is not clear yet.
