GB/T 40186-2021.Determination of genetic material damage strength for microbial mutation breeding-Umu method.
1 Scope
GB/T 40186 specifies the method for determining the strength of genetic material damage caused by microbial mutation breeding using the biological genetic toxicity (UMU) test method.
GB/T 40186 is applicable to the determination of the strength of genetic material damage caused by microbial mutation breeding using the UMU test method.
2 Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
mutagenesis
Inducing mutations in genetic material through artificial measures.
3.2
genetic material damage
Changes in the molecular structure of genetic material caused by chemical or physical mutagen.
3.3
SOS response
A stress response mechanism produced when DNA damage occurs in cells.
Note: In prokaryotes, it is mainly regulated by the SOS response system, which regulates the expression of more than 40 genes to cope with DNA damage.
3.4
​​ strength of genetic material damage
The degree of change in the molecular structure of genetic material caused by chemical or physical mutagen.
Note: When cells are damaged by DNA, emergency repair (SOS repair) will occur, allowing them to continue to survive at the cost of mutation. The strength of soS repair is often used
to represent the intensity of genetic material damage.
3.5
umu test methodumu test
A method in which the umuC gene encoding an important component of DNA polymerase V is fused with the lacZ gene encoding β-galactosidase and introduced into Salmonella typhimurium to determine the induction intensity of sos by detecting the activity of β-galactosidase.
Note: It can be used to determine the intensity of genetic material damage.
This standard specifies the method for determining the intensity of genetic material damage caused by microbial mutagenic breeding using the biological genetic toxicity (umu) test method. This standard is applicable to the determination of the intensity of genetic material damage caused by mutagenic breeding of microorganisms using the umu test method.

ICS07.080
National Standard of the People's Republic of China
GB/T40186—2021
Determination of genetic material damage strength for microbial mutationbreedingUmu method
Published on 2021-05-21
State Administration for Market Regulation
National Administration of Standardization
Implementation on 2021-12-01
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This standard was drafted in accordance with the rules given in GB/T1.12009. The standard was proposed and managed by the China National Institute of Standardization. GB/T40186—2021
Drafting units of this standard: Tsinghua University, Luoyang Huaqing Damu Biotechnology Co., Ltd., South China University of Technology, China National Institute of Standardization. The main drafters of this standard: Zhang Du, Xing Xinhui, Li Mei, Jian Xingjin, Wang Liyan, Guo Xiaojie, Zhang Lele, Li Shuang, Ma Aijin-riKaeerkca-
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1 Scope
Determination of the intensity of genetic material damage caused by microbial mutagenic breeding
Umu method
GB/T 40186-2021
This standard specifies the method for determining the intensity of genetic material damage caused by microbial mutagenic breeding using the biological genetic toxicity (umu) test method. This standard applies to the determination of the intensity of genetic material damage caused by microbial mutagenic breeding using the utnu test method. 2 Normative references
The following documents are indispensable for the use of this document. For all the referenced documents, the versions indicated are applicable to this document. For any undated referenced documents, the latest version (including all amendments) shall apply to this document GB/T 6682 Specifications and test methods for water used in analytical laboratory air 3 Terms and definitions || tt || The following terms and definitions shall apply to this document.
Mutagenesis
Induce mutations in genetic material through artificial measures. 3.2
Genetic material damage genetic material damage Changes in the molecular structure of genetic material caused by chemical or physical mutagens: 3.3
Emergency response Sos response
A stress response mechanism produced when deoxyribonucleic acid (DNA) is damaged in cells: Note: In prokaryotes, it is mainly regulated by the stress response (SOS response) system, which regulates the expression of more than 40 genes to cope with DNA damage. 3.4
Strength of genetic material damage Chemical or physical mutagens divide the degree of structural changes in genetic material into small and large ones. Note: When a cell is damaged by DNA, emergency repair (SOS repair) will occur, allowing it to continue to survive at the cost of mutation. The strength of SOS repair usually represents the strength of genetic material damage.
umu test
umu test
A method in which the umuC gene encoding an important component of DNA polymerase V is fused with the lacZ gene encoding 3-galactosidase and introduced into Salmonella typhimurium. The induction intensity of SOS is determined by detecting the activity of 3-galactosidase.
Note: It can be used to determine the intensity of genetic material damage 1
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GB/T40186—2021www.bzxz.net
4 Principle
Umu test method, using fluorescein-2-3-D-galactoside as the fluorescent substrate of 3-galactosidase, using iodinated endopyrin as a dead cell dye, and using flow cytometry fluorescence sorting technology (FACS) to determine the 3-galactosidase activity of living cells after mutagenesis, thereby obtaining the genetic material damage intensity of living cells. 5 Reagents or materials
Unless otherwise specified, analytically pure reagents shall be used: water shall be grade-1 water as specified in GB/T6682. 5.11 mol/L hydrochloric acid solution
Measure 83.3 mL of 12 mol/L concentrated hydrochloric acid and dissolve it in water to make the volume 1 L to obtain a hydrochloric acid solution with a concentration of 1 mol/L. 5.21mol/L sodium hydroxide solution
Weigh 40.0g sodium hydroxide and dissolve it in water to make up to 1L5.3 Test microbial culture medium (TGA)
Weigh 10.0g trypsin, 5.0g sodium chloride and 11.9g 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), add 980mL water to dissolve. Adjust the pH to 7.0±0.2. Make up to 1000mL. 121℃. 15min high pressure sterilization: dissolve 2.0g grape chrysanthemum in 20mL deionized water and sterilize separately. After sterilization, mix the two solutions in equal proportions. Add 50.00mg ampicillin per liter of cooled TGA culture medium under sterile conditions. The solution can be stored at -20℃ for 4 weeks. 5.4 Fluoresceindi-β-D-galactopyranoside (FDG) solution: Dissolve 13.13 mg FLDG in 10 mL of water containing 1% DMSO and 1% ethanol by volume. The final concentration of FDG is 2 mmol/L. Store in a refrigerator at -20℃ after aliquoting. Preheat at 37℃ for more than 15 minutes before use. 5.5 Propidium iodide dide.Pl) solution Dissolve 6.68mgPI in 10mLPBS solution. Prepare PI solution with a concentration of 1mmol/L: Use a pipette to draw 10uL of PI stock solution with a concentration of 1mmol/L, dissolve it in 10mLPBS solution, and prepare PI solution with a concentration of 1mol/L. Store it at 4℃ as PI working solution, and place it on ice for precooling before use6Instruments
Constant temperature water bath: temperature can reach 37℃1℃6.2pII meter, accuracy 0.1
Electronic balance, accuracy 0.01mg.
High-speed centrifuge.
6.5Flow cytometer.
Constant temperature shaking bed. The temperature can reach 37℃±1℃, and the speed range meets 125r/min~250r/min. 6.7
Ultraviolet-visible spectrophotometer, with a detectable wavelength of 600nm±20nm, equipped with 1cm colorimetric III: -riKacerKAca-
7Sample
7.1 Test microorganisms
GB/T40186—2021
Salmonella typhimurium VM20o9. Plasmid containing idarumuC-lucZ gene and aminocyanocin resistance gene (see Appendix A for plasmid sequence). 7.2 Storage of test microorganisms
Put 150 μL of the test microorganism culture in a cryopreservation tube, add 350uL of an aqueous solution containing 10% (volume fraction) disulfoxide or 20% (volume fraction) glycerol, mix thoroughly, and store in a refrigerator at -80℃. 8 Test steps
8.1 Overnight culture of test microorganisms
The process of overnight culture of test microorganisms is as follows: a) Fill a 100mL shaking flask with 20mL TGA medium, seal it with a breathable stopper, and store it in a refrigerator: h) Thaw the frozen test microorganisms at room temperature. Then add 1ml of TGA medium to the cryopreservation tube: c)
Centrifuge the test microorganisms in the cryopreservation tube at 40001/min for 10min. Then discard the supernatant and resuspend the test bacteria with 1ml TGA medium;
cl) Inoculate 0.5mL of the test microorganism resuspension into the shaking flask containing TGA medium and culture it in a 37℃+1℃ incubator overnight (no more than 12h).
8.2 Prepare the mutagenic sample and control sample of the test microorganism. Dilute the test microorganism cultured overnight by 10 times with fresh TGA medium, continue to shake and culture in a constant temperature shaker at 37°C±1°C, and set the detection wavelength to 600nm-20nm. Use TGA medium as a blank control, and use 1cm colorimetric III to detect whether the test microorganism sample is in the logarithmic growth phase after culture. Mutagenic groups are obtained by mutagenic treatment of the test microorganism sample by physical mutagenesis or chemical mutagenesis. Control samples are obtained by non-mutagenic treatment. The amount of mutagenic sample of the test microorganism should be 200ml. Transfer the mutagenic experimental samples and control groups to 1.5ml centrifuge tubes and culture them in a 37-1°C shaking incubator at 200r/min for 2h8.3IFDG and PI staining
Take 50L of the test samples of the mutagenesis group and the control group and place them in a 1.5mL centrifuge tube. After preheating at 37℃ for 5min, add 50μL of FDG solution preheated at 37℃. Mix quickly and gently and place in a 37℃ water bath for 2min to allow FDG to penetrate into the cells: then quickly add 500L of PI solution to the above 1.5mL centrifuge tube, and place the mixture on ice for 1h. Keep it on ice before flow cytometry. 8.4 Flow cytometer determination
Flow cytometer is used to analyze the stained samples: set the excitation wavelength to 488nm. For the fluorescence intensity measurement of the FDG hydrolysis product fluorescent cord, the receiving wavelength is 525nm (FIl-1 channel). The receiving wavelength for PI staining fluorescence measurement is 670nm (Fl-2 channel). The total number of cells to be measured is set to 5 000~100008.5Threshold setting
Circle the target bacteria by forward scattered light FSC and side scattered light SSC to remove the influence of multi-cell adhesion results. 3
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GB/T40186—2021
By single PI staining, measure the relative fluorescence estimation of FL1 and FL2 channels. Circle the PI stained cells on the FL1 axis. The relative fluorescence value of FI-1 channel of the circled cells has a range. The cells with FL-I relative fluorescence value not less than this range are PI staining negative cells
By single FDG staining, measure the relative fluorescence value of FL-1 and FL-2 channels. Circle the FDG stained cells on the FL-2 axis. The relative fluorescence value of FL2 channel of the circled cells has a range.The cells within the range of FL2 relative fluorescence value are FDG-positive cells. For samples stained with FDG and PI at the same time, cells with positive FDG staining and negative PI staining are selected as the target cell population for subsequent data processing and result analysis. Experimental data processing SOs induction coefficient is calculated by formula (1): Where: SOS induction coefficient: A. ——The average FDG hydrolysis product fluorescein relative fluorescence value of samples treated with mutagenic source; A nr ——The average FDG hydrolysis product fluorescein relative fluorescence value of control samples: The arithmetic mean of the results of two parallel samples is reported, and the results are retained to the decimal place. Appendix A (Informative Appendix) Plasmid sequence information GB/T40186—2021
The high-quality sequence information of Salmonella typhimurium VM2009, which contains the expression of umuC-lucZ gene and aminocyanide resistance gene, is as follows:
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GB/T40186—2021

GB/T40186—2021

GB/T 40186-2021
People's Republic of China
National Standard
Determination of the Intensity of Damage to Genetic Material Caused by Microbial Mutation BreedingUmu Method
GB/T4C1862021
Published and distributed by China Standards Press
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Service Hotline: 4001680010
First edition in May 2021
Book number: 155066: 1-63953
Copyright reserved
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Infringements will be investigatedcn
Service hotline: 4001680010
First edition in May 2021
Book number: 155066: 1-63953
Copyright reserved
-rrKaeerkAca-
Infringement will be investigatedcn
Service hotline: 4001680010
First edition in May 2021
Book number: 155066: 1-63953
Copyright reserved
-rrKaeerkAca-
Infringement will be investigated
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