Team:XJTU-China/Measurement
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Measurement
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Overview
During the development of the project, we sorted out and tested the general methods for the determination of polysaccharide content in the culture medium. We also designed quantitative methods for the determination of available phosphorus content, total nitrogen content, organic matter content, and various inorganic salt ion content in the soil.
For the determination of polysaccharide content, we used a determination method that uniformly converted the polysaccharide in culture medium into glucose equivalent, which not only ensured certain determination accuracy, but also avoided the tedious operation of polysaccharide purification, multiple separation, hydrolysis and re-separation.The phenol sulfuric acid method for determination of glucose content was also applied in the experiment to determine the ability of engineering bacteria to produce EXTRA Cellular polysaccharides.
For the determination of soil-related parameters, we have integrated a variety of methods, such as the molybdenum-antibiotic colorimetric method, to determine the content of available phosphorus in soil.Our method has been used to determine the available phosphorus content and other parameters of soil samples collected from several desert areas such as Yulin Cherishing sand plant protection base, which basically proves the feasibility of the method.
For the determination of polysaccharide content, we used a determination method that uniformly converted the polysaccharide in culture medium into glucose equivalent, which not only ensured certain determination accuracy, but also avoided the tedious operation of polysaccharide purification, multiple separation, hydrolysis and re-separation.The phenol sulfuric acid method for determination of glucose content was also applied in the experiment to determine the ability of engineering bacteria to produce EXTRA Cellular polysaccharides.
For the determination of soil-related parameters, we have integrated a variety of methods, such as the molybdenum-antibiotic colorimetric method, to determine the content of available phosphorus in soil.Our method has been used to determine the available phosphorus content and other parameters of soil samples collected from several desert areas such as Yulin Cherishing sand plant protection base, which basically proves the feasibility of the method.
Soil property measurement
To understand the possible problems in the practical application of the project, we carried out soil experiments to measure the physical and chemical properties of the pre-applied soil. It is a necessary step to predict the sustainable development ability and application range of the system by cooperating with the modeling part in soil experiments.
A considerable part of our measurement focuses on the measurement of environmental indicators. After the previous field sampling, in the laboratory, we measured the soil water content, organic matter, available phosphorus, total nitrogen, total sodium, PH, and density. This part of the data will serve for the model and future culture experiments.
A considerable part of our measurement focuses on the measurement of environmental indicators. After the previous field sampling, in the laboratory, we measured the soil water content, organic matter, available phosphorus, total nitrogen, total sodium, PH, and density. This part of the data will serve for the model and future culture experiments.
Sampling
In obtaining soil samples, we have been helped by many parties to obtain four groups of samples of sandy land distributed in four provinces in China:
1.Alashan, Inner Mongolia, N39°33’11”, E102°21’24” H1168m;
2.The management and protection station in Kalamaili Mountain ungulate Nature Reserve, Xinjiang,N44°45’18.66”E88°49’8.29” H460m
3.Yulin city, Shaanxi province, N37°31’58.66”，E109°0’1.62”;
4.Zhangye, Gansu province, N38°56′,E100°26′
1.Alashan, Inner Mongolia, N39°33’11”, E102°21’24” H1168m;
2.The management and protection station in Kalamaili Mountain ungulate Nature Reserve, Xinjiang,N44°45’18.66”E88°49’8.29” H460m
3.Yulin city, Shaanxi province, N37°31’58.66”，E109°0’1.62”;
4.Zhangye, Gansu province, N38°56′,E100°26′
The collection of soil samples follows the professional collection method, and samples with 0-5cm of soil surface are collected, sealed and packaged, and then sent to the laboratory for subsequent experiments.
Method of measurement
The measurement of soil index refers to the Chinese national standard and iso standard, and appropriate adjustments are made according to laboratory conditions. All measurements are made after the soil is sieved. With the help of the engineer Yu Zhang from the environmental engineering laboratory, we have designed the following complete measurement protocol:
Read Pdf
https://2020.igem.org/File:T--XJTU-China--Measurement1pdf.pdf
Measuring result
| unit | Alashan-Bare sand | Alashan-Vegetation | Alashan-crust | Yulin | Xinjiang | Zhangye 0-5cm | Zhangye 5-10cm | |
|---|---|---|---|---|---|---|---|---|
| water content | 0.25% | 0.42% | 0.65% | 1.00% | 0.97% | 0.49% | 1.69% | |
| organic matter | 0.72% | 2.35% | 1.45% | 1.40% | 0.93% | 1.25% | 1.33% | |
| available phosphorus | mg/kg | 0.888 | 3.676 | 7.068 | 1.090 | 6.389 | 8.915 | 8.544 |
| total nitrogen | 0.026% | 0.028% | 0.030% | 0.003% | 0.027% | NA | NA | |
| total sodium | g/kg | 1.142 | 1.327 | – | – | – | – | – |
| PH | 5.66 | 7.05 | 10.09 | 9.16 | 8.42 | 8.82 | 9.14 | |
| density | g/L | 1.426 | 1.446 | 1.307 | 1.343 | 1.170 | 1.364 | 1.263 |
Analysis
Soil water content
Soil water pattern is often the decisive factor affecting the settlement and growth of pioneer species in the early succession of desert ecosystem. Soil biological crust significantly changes the infiltration process of precipitation and the redistribution of soil water, slows down the occurrence of surface runoff or soil erosion, and affects surface evaporation, thus increasing the water content of surface soil.
The research shows that the water content of the sand layer in the desert area is low, and the water content of the sand layer above 0.2m depth is usually less than 1%. With the increase of soil depth, the soil water content generally showed an upward trend. Our experimental results also accord with this conclusion. The water content of 5-10cm soil samples in Gansu is significantly higher than that of 0-5cm soil samples.
The research shows that the water content of the sand layer in the desert area is low, and the water content of the sand layer above 0.2m depth is usually less than 1%. With the increase of soil depth, the soil water content generally showed an upward trend. Our experimental results also accord with this conclusion. The water content of 5-10cm soil samples in Gansu is significantly higher than that of 0-5cm soil samples.
Soil organic matter
Soil organic matter affects various physical and chemical properties of soil, and its content determines the growth status and diversity of surface plants. At the same time, the change of soil organic matter content is an important index to evaluate whether the land is desertification or not.
Current research shows that the content of soil organic matter in the 0-60 cm soil layer is a regional variable with random distribution, and spatial autocorrelation does not exist, that is, it is significantly affected by external factors, including natural and man-made factors. From our data, we can see that the distribution of organic matter content in samples is about 1%. Soil with vegetation obviously has higher organic matter content, while bare sand has the lowest organic matter content.
Current research shows that the content of soil organic matter in the 0-60 cm soil layer is a regional variable with random distribution, and spatial autocorrelation does not exist, that is, it is significantly affected by external factors, including natural and man-made factors. From our data, we can see that the distribution of organic matter content in samples is about 1%. Soil with vegetation obviously has higher organic matter content, while bare sand has the lowest organic matter content.
Soil available phosphorus
Dissolved inorganic phosphate is the main source of P for microorganisms and plants, but its availability in soil, sediment, and water is very low due to its high reactivity with calcium, iron, and aluminum. Because the primary productivity and growth rate of the ecosystem is highly dependent on P, microorganisms have evolved many mechanisms to absorb and store P in response to nutrient deficiency. The phosphorus content in desert particles is very small, and only a part of them can participate in the biological cycle, that is, they can be transformed into bioavailable forms through physical, chemical, and biological processes, which are collectively called bioavailable phosphorus.
According to the literature, the available phosphorus in desert soil is usually 2-6mg/kg. Although our results are not perfectly consistent, they are around this range. The available phosphorus of different samples is quite different. The content of available phosphorus in bare sand is the lowest, and in the case of vegetation, the content of available phosphorus decreases, which is presumed to be absorbed by plants.
According to the literature, the available phosphorus in desert soil is usually 2-6mg/kg. Although our results are not perfectly consistent, they are around this range. The available phosphorus of different samples is quite different. The content of available phosphorus in bare sand is the lowest, and in the case of vegetation, the content of available phosphorus decreases, which is presumed to be absorbed by plants.
Soil total nitrogen
The biological soil crust has fixed more than 40% of the global biological nitrogen with 7% primary productivity, but its nitrogenase activity is more affected by its damage degree. The nitrogenase activity of biological crust decreases by 30%–100% due to interference, which has an important impact on the nitrogen budget of the desert ecosystem. Soil nitrogen includes organic nitrogen, ammonium nitrogen, nitrate-nitrogen, and nitrite nitrogen. Organic nitrogen can generally account for more than 95% of total nitrogen; Nitrite nitrogen is unstable and its content is very small.
According to previous studies, the total nitrogen content in desert soil is generally 0.1-0.4mg/g, and our results are partially satisfying. However, it can be found that the soil nitrogen content is extremely low, which deserves attention and may become a difficult point in the application process.However, due to the limitation of conditions, nitrate nitrogen is not included in our detection, although it has no significant effect on total nitrogen.
According to previous studies, the total nitrogen content in desert soil is generally 0.1-0.4mg/g, and our results are partially satisfying. However, it can be found that the soil nitrogen content is extremely low, which deserves attention and may become a difficult point in the application process.However, due to the limitation of conditions, nitrate nitrogen is not included in our detection, although it has no significant effect on total nitrogen.
Soil total sodium and PH
As for conditions, only some samples were tested for soil sodium. Compared with the current literature, our data have little difference. Na content in soil is used to understand inorganic salt levels in the soil. Soil PH varies greatly, with strong heterogeneity, and most of them are weakly alkaline.
Summary
Reliability
The measurement of soil property refers to the Chinese national standard and ISO standard, which can be repeated by other iGEM teams as an accurate method for measuring environmental index. In the experimental design, blank control groups were set for all samples to reduce possible errors. During the experimental measurement, multiple parallel experiments were set for each group of samples to test the feasibility and reproducibility of the experimental method.
Availability
The detailed protocol is presented in the PFD file for other iGEM teams to consult and use. Our measurement method has a wide range of applications for the determination of soil properties and has a useful value for many teams applying engineering bacteria to the soil environment. For many environmental track events, it is very important to understand the environment to clarify its relationship with microorganisms. Our measurement method is the reflection of interdisciplinary hot spots.
Polysaccharide measurement
Overview
Athrone colorimetry was mainly used in the detection of polysaccharides. A series of compounds, such as furfural, can be formed by dehydration of sugar by sulfuric acid. The concentration of polysaccharide can be determined by measuring the absorbance at 620nm.
Method of measurement
Read Pdf
https://2020.igem.org/File:T--XJTU-China--Measurement2pdf.pdf
Feasibility and reproducibility
We attached great importance to the reliability of polysaccharide detection in feasibility and reproducibility, and consulted professors in relevant fields to obtain relevant feedback, and improved the experimental process according to the feedback.
At first, the colorimetric method was to use ultraviolet absorption spectrophotometer to measure the absorbance at 620nm. However, due to different models, different operation methods, and the method itself has a wider demand for the absorption range, we replaced the colorimetric instrument from UV-Vis spectrophotometer to enzyme labeled instrument, and used 96-well plate for one-time measurement, which avoided the error caused by operation and significantly improved the feasibility and reproducibility.
At first, the colorimetric method was to use ultraviolet absorption spectrophotometer to measure the absorbance at 620nm. However, due to different models, different operation methods, and the method itself has a wider demand for the absorption range, we replaced the colorimetric instrument from UV-Vis spectrophotometer to enzyme labeled instrument, and used 96-well plate for one-time measurement, which avoided the error caused by operation and significantly improved the feasibility and reproducibility.
Visualization and Statistical Analysis
In addition, the use of microplate has significant advantages in the use of appropriate data visualization, statistical analysis and units. By using the colorimetric results of the enzyme labeled instrument, the data can be directly exported to the excel document, and further data processing can be carried out in the excel document. The excel document is directly saved in the offline working computer connected with the enzyme marker to ensure the file security. This method meets the requirements of visualization, unit and easy statistical analysis of experimental data.
Data processing and analysis
The results are divided into two parts. The first part is the drawing of glucose standard curve. The glucose concentration under different gradients can be measured by microplate, and the standard curve of the relationship between glucose concentration and absorbance can be calculated. The standard curve obtained is y (absorbance) = 0.5562x (mmol / L) + 0.0592, R2 = 0.997, the linear degree is good, this relationship can be used to calculate the concentration of polysaccharide.
The second part is the determination of polysaccharide content. The results show that the absorbance of dh5a in the control group is 0.3467, and that of BB, be, BP, EB, EE and EP in the experimental group is 0.4322, 0.4575, 0.4369, 0.5073, 0.6563, 0.5267, respectively. The absorbance of all experimental groups is higher than that of the control group The results showed that PGMA gene and galU gene of E. coli had more significant effect on polysaccharide overexpression.
Read Pdf
https://2020.igem.org/File:T--XJTU-China--protocol_-2.pdf