Team:NAU-CHINA/Contribution

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Contributions to an Existing Part

BBa_K525515

We find that there’s no information about “LC-ESI-qTOF-MS(-MS)”, which is a method to detect the pollutant of BPA.

But we have found the similar method “LC-MS/MS”. This method uses "methyl alcohol - water" as the fluid phase, separated by the Waters ACQUITY UPLC HSS T3 chromatographic column separation and determined by the UPLC/MS/MS quantitative assay. This method has high repeatability and precision, and can meet the requirements of the BPA reference value limit determined in the "Drinking Water Hygiene Standard" (GB 5749-2006).

Here're the results of the research：

Table1.Linear equation, correlation coefficient, LOD and LOQ of BPS, BPF and BPA in pure water(Zhou Xiaoxin, et al., Journal of Environmental Hygiene. 2020)

Compound Linear equation Correlation coefficient LOD（μg/L） LOQ（μg/L）
BPS y=3.11×106x-4.4×105 0.9999 0.002 0.007
BPF y=5.22×104x-5.2×104 0.9974 0.05 0.17
BPA y=1.87×104x+1.64×104 0.9996 0.15 0.50

According to Table1, the standard curve is plotted with the concentration of the standard series as the x-coordinate and the peak area of the corresponding compound as the vertical coordinate. BPS, BPF and BPA show good linearity in the range of (0.5 ~ 50.0) g/L and their correlation coefficients are bigger than 0.997.

Table2. Precision and recovery of BPS, BPF and BPA in different water samples (N = 6)(Zhou Xiaoxin, et al., Journal of Environmental Hygiene. 2020)

Sample name Compound Background/(μg/L) Added Scalar/(μg/L) Amount recycled/(μg/L) Average recovery rate/% RSD/%
2.00 1.91~2.05 100 3.19
Pure water BPS Not detected 7.50 7.26~7.77 101 2.95
25.0 22.9~26.8 103 5.90
2.00 1.91~2.06 98.4 2.85
BPF Not detected 7.50 7.67~7.79 103 0.65
25.0 24.1~25.5 98.7 2.44
2.00 1.93~2.09 101 3.09
BPA Not detected 7.50 7.33~7.51 99.1 0.85
25.0 23.7~25.1 98.1 2.06
2.00 2.07~2.20 108 2.31
Spring water BPS Not detected 7.50 7.13~7.30 96.4 1.03
25.0 21.6~22.5 88.3 1.71
2.00 1.93~2.00 98.3 1.47
BPF Not detected 7.50 7.23~7.50 98.7 1.37
25.0 21.8~24.1 92.4 3.57
2.00 2.03~2.06 102 0.57
BPA Not detected 7.50 7.33~7.51 99.2 0.85
25.0 24.1~26.9 102 4.03
2.00 1.96~2.08 102 2.46
Tap water BPS Not detected 7.50 6.57~6.95 90.1 2.35
25.0 23.3~24.4 95.8 1.92
2.00 1.90~1.99 97.3 1.65
BPF Not detected 7.50 7.36~7.53 99.2 0.98
25.0 22.9~25.5 99.1 4.11
2.00 1.96~2.05 99.3 1.71
BPA Not detected 7.50 7.34~7.56 98.8 1.05
25.0 22.7~25.2 96.0 3.92

This study analyzed 5 parts of pure water, 5 parts of mineral water, 1 part of distilled water and 10 parts of terminal water respectively. The results showed that BPS, BPF and BPA were not detected in 11 commercially available samples and 6 terminal water samples but BPA was detected in 4 parts of terminal water with concentrations of (0.59 ~ 5.23) g/L.

Thus, “LC-MS/MS” has the advantages of strong qualitative ability, strong stability and high sensitivity, and is often used in the determination of bisphenol compounds.

BBa_K1067006

The problem is that N2O couldn’t be detected precisely during the denitrification reaction, but we have discovered a new method.

Fig.1. Schematic diagram of SBR device(Liu Guohua, et al.,Environmental Engineering. 2020)

The gas samples are collected by the portable gas sampling pump every 30 min within one operating cycle and collected in a 1L gas sampling bag. The concentration of N2O was determined by Agilent 6890N gas chromatography (GC) equipped with ECD detector.

The N2O accumulation released in aerobic stage was 8.2 mg, about 6 times of that released in anoxic stage. According to the calculation, the conversion of N2O occurs mainly in the aerobic stage, and its conversion rate was 2.84%.

Fig.2. N2O emission rates and N2O cumulative emission in A/O- SBR system(Liu Guohua, et al., Environmental Engineering. 2020)

This study got some conclusions:

①The release of N2O occured mainly in the aerobic stage of A/O-SBR sewage treatment pilot system.

②The maximum release rate of N2O was 2.02 μgmin-1 gmLSS-1 during one operating cycle of the A/O-SBR small scale test system. The cumulative release rate reached 8.2 mg after 300 min of operation, and the accumulation of nitrite also reached the maximum value of 7.5 mg/L.

Although the equipment is a little big to use in the laboratory, we can improve it in the future.

BBa_K1096001

We checked the data of gene mazE, Part:BBa_K1096001, finding that there was no data about using CRISPRi technology to knock down the gene mazEF of E. coli Nissle1917 in situ and exploring the relevant data about its impact on biofilm and persistence. Thus, we checked the literature to supplement it.

Knock down the mazEF gene and hipA gene of E. coli Nissle1917 in situ

The system of mazE and hipA was successfully knocked down through RT-PCR verification, and the expression of other gene clusters was not affected.

Knock down mazEF gene and hipA gene in situ to explore its impact on persister bacteria and biofilm

This experiment used CRISPRi to knock down the mazEF gene and hipA gene in situ in EcN1917 to study the influence of mazEF gene and hipA gene on the formation of biofilm and persister bacteria.

①Determination about the effect of mazE on biofilm

Experimental method: crystal violet staining method (quantitative)

Fig.3. Comparison chart of relative biofilm of each strain(Li Pinyi. Zhejiang Gongshang University. 2014)

Original plasmid had no significant effect on growth. But after CRISPRi knockdown expression, the biofilm formation of EcN-ΔmazE and EcN-ΔhipA was significantly inhibited.

②Determination about the effect of mazE on persister bacteria

They used a new method to test persistence — enzyme lysis method to avoid resistance cross-interference. They also adopted the classic method of persistence formation determination, the antibiotic method, to repeat the sterilization curve determination for verifying TA expression’s influence on strain’s persistence.

Fig.4. The killing curve on effect of mazEF and hipA on the formation of persiser cell(Li Pinyi. Zhejiang Gongshang University. 2014)

This figure shows that the knockdown of mazE and hipA genes inhibits the formation of persister bacteria.

3D Model

Summary

In order to better and more comprehensive understand our favorite reporter whose name is BBa_E1010, this year NAU-CHINA uses SWISS MODEL (https://swissmodel.expasy.org/)to model and simulates the tertiary structure of protein. We hope that the prediction of the structure will help other teams to better understand the nature and characteristics of this part and be able to use the reporter gene more skillfully.

The following model was built (see Materials and Methods "Model Building"):

Fig.5. Model #01

Fig.6. the Active center

Fig.7. Local Quality Estimate

Fig.8. Comparison with Non-redundant Set of PBD Structures

Codon Optimization

Optimized CⅠ(BBa_K3408004)

We optimized the CⅠ protein (BBa_K3408004) based on ThermoFisher SCIENTIFIC to make it more suitable for our engineered bacteria.

Fig.9. Codon quality distribution

Fig.10. Optimized codon quality distribution

The histograms show the percentage of sequence codons which fall into a certain quality class. The quality value of the most frequently used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly (Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3),1987).

Optimized DpnI(BBa_K3408005)

Bacillus subtilis has been increasingly applied in genetic engineering due to its powerful secretion capacity. As a restriction enzyme which is capable of cutting all methylated DNA, optimized DpnI (BBa_K3408005) via codon optimization can be applied in Bacillus subtilis more efficiently.

Fig.11. Codon quality distribution

Fig.12. Optimized codon quality distribution

The histograms show the percentage of sequence codons which fall into a certain quality class. The quality value of the most frequently used codon for a given amino acid in the desired expression system is set to 100, the remaining codons are scaled accordingly (Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3),1987).

References