Team:NAU-CHINA/Contribution text

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BBa_K 525515

We find that there’s no information about “LC-ESI-qTOF-MS(-MS)”, which is a method to detect the pollutant of BPA[1].

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. The method could meet the requirement for the determination of BPA reference value limit specified in the Sanitary Standard for Drinking Water (GB 5749-2006), with high reproducibility and precision.

Here're the results of the research：

Table.1. 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（mg/L） LOQ（mg/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 Table 1, 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.

Table.2. 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/8mg/L9 Scalar Added/8mg/L9 Amount recycled MN/8mg/L9 Average recovery rate/% RSD/%
BPS
Pure water 2.00 1.91~2.05 100 3.19
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
Spring water 2.00 2.07~2.20 108 2.31
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
Tap water 2.00 1.96~2.08 102 2.46
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_K 1067006

A 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 1 L 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 is 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 occurs 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 300min 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.

Knockdown 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-▲mazE and EcN-▲hipA 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 simulate 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"):

Fig1. Model #01

Fig.2 the Active center

Fig.3local quality

Fig.4 Comparison with Non-redundant Set of PBD Structures

Codon optimization

Optimized CI(BBa_K3408004)

We have optimized the CI protein on ThermoFisher SCIENTIFIC(https://www.thermofishe r.com/cn/zh/home/life-science/cloning/gene-synthesis/geneart-gene-synthesis/geneoptimizer.html)to use it better in our engineering bacteria.

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 (see also Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3),1987).

Optimized DPNI(BBa_K3408005)

Bacillus subminis 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, DPNI can be applied to Bacillus subtilis in the way of synthetic biology to control the environment by taking advantage of its DNA eradication.

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 (see also Sharp, P.M., Li, W.H., Nucleic Acids Res. 15 (3),1987).

References