Team:XHD-Wuhan-A-China/Implementation
Proposed Implementation
Introduction
Rhizobium exist widely in the soil environment and directly participate in the nitrogen cycle. They can not only reduce nitrogen in the air to ammonia that can be used by plants, but also have the ability to convert the combined nitrogen back into nitrogen, a process called denitrification. At present, there are many researches on nitrogen fixation of rhizobium, but few researches on denitrification of rhizobium, among which the potential application direction is worth further exploring. With the process of human industrialization, the excessive use of chemical fertilizers leads to the destruction of soil ecological environment, which is directly manifested as soil salinization. In particular, the accumulation of nitrate often leads to soil acidification and compaction. Therefore, our project design is to use rhizobium to degrade excessive nitrate in the soil to solve the soil compaction caused by soil salinization.
According to the experimental procedure designed by our project, we linked napA and nirK genes to pigment protein amilCP, respectively, to evaluate the copy number of additional imported napA and nirK genes. We used machine learning model to predict and mutate the Pyear promoter to enhance the strength of the promoter, which laid a foundation for the reasonable perception of external nitrate concentration in rhizobia. Denitrifying enhanced rhizobia can be used in the following areas:
Application to improve soil compaction
As the material basis for human survival, the ecological situation of soil is not optimistic in recent years and its quality is deteriorating day by day. But people pay far less attention to it. Irrational cultivation aggravates the degradation of land, especially the excessive use of chemical fertilizers, resulting in the increase of soil nitrate content, and damage to the soil environment. Soil compaction will increase soil hardness, resulting in a decrease in soil water storage, water retention and water conductivity, and ultimately lead to more fertilizer inputs. Excessively hard soil is also bad for plants to absorb fertilizer, further worsening the level of soil salinization. Through our rhizobium, the nitrate in the soil can be decomposed, reducing the content of salt ions in the soil, so as to solve the soil compaction.
Keep the soil at a reasonable nitrate concentration
High nitrate concentrations in the soil can lead to soil compaction, and plants cannot thrive in nitrogen-deficient soils. Therefore, it is important to maintain a normal concentration of nitrate ions in the soil. We studied the Pyear promoter, which is the promoter of the Escherichia coli yeaR/yoaG operon and, unlike other Escherichia coli promoters that respond to nitrate and nitrite, is not inhibited under aerobic conditions. We performed promoter strength modelling by means of machine learning and mutated the Pyear promoter to enhance Pyear promoter strength. This promoter allowed rhizobium to reasonably sense the nitrate concentration in the environment and thus exerted a reasonable intensity of denitrification.
The new iGEM chassis creature
In iGEM projects over the years, Escherichia coli was generally used as chassis creature in many projects. However, there are few projects using rhizobium as chassis creature, mainly because of the slow growth rate and low transformation efficiency of rhizobium. As the participants of nitrogen cycle in nature, rhizobium inherently have a dual function of nitrogen fixation and denitrification and are natural chassis creature designed to be involved in the nitrogen cycle circuit. We made a preliminary design of a suitable gene expression system for rhizobium, which provided a reference for related studies.
Possible problems and challenges
The genetic background of Sinorhizobium fredii HH103 is not as well studied as that of E. coli, so various unexpected problems may be encountered in the experiments. Moreover, the growth cycle of Sinorhizobium fredii HH103 is longer than that of Escherichia coli. So the culture time and experimental process are more time-consuming. In this project, we completed most of the experiments. However, it is not clear whether the denitrification-enhanced Sinorhizobium fredii HH103 is effective in the natural environment. While performing synthetic biology with Sinorhizobium fredii HH103 as a biological chassis, the following problems were identified.
1. Sinorhizobium fredii HH103 plasmid transformation efficiency is relatively low, which will make the experimental cycle uncontrollable.
2. Sinorhizobium fredii HH103 is a fast-growing soybean rhizobium, but the growth rate is still not as high as that of E. coli, resulting in a longer overall experimental cycle.
3. The currently used plasmid skeleton has a low copy in rhizobium, resulting in low gene expression.
References
Margaret, I. , Lucas, M. M. , Sebastián Acosta-Jurado, Ana M. Buendía-Clavería, Fedorova, E. , & Ángeles Hidalgo, et al. (2013). The sinorhizobium fredii hh103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules. PLoS ONE, 8(10), e74717.
Ouni Y , Albacete A, Cantero E, et al. Influence of municipal solid waste (MSW) compost on hormonal status and biomass partitioning in two forage species growing under saline soil conditions. Ecological Engineering,2014,64:142-150.
Vinardell, J. M. , Acosta-Jurado, S. , Gttfert, M. , Zehner, S. , & Weidner, S. . (2015). The sinorhizobium fredii hh103 genome: a comparative analysis with s. fredii strains differing in their symbiotic behavior with soybean. Mol Plant Microbe Interact, 28(7), 811-824.
