GB/T 40186-2021 PDF in English
GB/T 40186-2021 (GB/T40186-2021, GBT 40186-2021, GBT40186-2021)
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Determination of genetic material damage strength for microbial mutation breeding - Umu method
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GB/T 40186-2021: PDF in English (GBT 40186-2021) GB/T 40186-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 07.080
A 21
Determination of Genetic Material Damage Strength
for Microbial Mutation Breeding - Umu Method
ISSUED ON: MAY 21, 2021
IMPLEMENTED ON: DECEMBER 1, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Terms and Definitions ... 4
4 Principle ... 5
5 Reagents or Materials ... 5
6 Instruments and Equipment ... 6
7 Samples ... 6
8 Test Procedures ... 7
9 Test Data Processing ... 8
Appendix A (informative) Plasmid Sequence Information ... 10
Determination of Genetic Material Damage Strength
for Microbial Mutation Breeding - Umu Method
1 Scope
This Standard specifies the method for determining genetic material damage strength
for microbial mutation breeding through the biogenic toxicity (umu) test method.
This Standard is applicable to the determination of genetic material damage strength
for microbial mutation breeding through the umu test method.
2 Normative References
The following documents are indispensable to the application of this document. In
terms of references with a specified date, only versions with a specified date are
applicable to this document. In terms of references without a specified date, the latest
version (including all the modifications) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
3 Terms and Definitions
The following terms and definitions are applicable to this document.
3.1 Mutagenesis
Mutagenesis refers to the induction of mutations in genetic material through man-made
measures.
3.2 Genetic Material Damage
Genetic material damage refers to changes in the molecular structure of genetic
material by chemical or physical mutagenesis sources.
3.3 SOS Response
SOS response refers to an emergency response mechanism generated when cells
manifest deoxyribonucleic acid (DNA) damage.
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.
the pH to 7.0 ± 0.2; reach a constant volume of 1,000 mL. At 121 °C, autoclave it for
15 min. Dissolve 2.0 g of glucose in 20 mL of deionized water; separately sterilize it.
After sterilization, in equal proportions, mix the two solutions. Under aseptic conditions,
add 50.00 mg of ampicillin per liter of cooled TGA medium. This solution can be stored
at -20 °C for 4 weeks.
5.4 Fluorescein di--D-galactopyranoside (FDG) Solution
In 10 mL of water containing 1% (volume fraction) dimethyl sulfoxide (DMSO) and 1%
ethanol, dissolve 13.13 mg of FDG. The final concentration of FDG is 2 mmol/L. After
aliquoting, store in the refrigerator at -20 °C. Before use, at 37 °C, pre-heat it for more
than 15 min.
5.5 Propidium Iodide (PI) Solution
In 10 mL of PBS solution, dissolve 6.68 mg of PI to prepare a PI solution with a
concentration of 1 mmol/L. Use a pipette to draw 10 μL of PI stock solution with a
concentration of 1 mmol/L; dissolve it in 10 mL of PBS solution to prepare a PI solution
with a concentration of 1 μmol/L; use it as PI working solution. Store it at 4 °C. Before
use, place it on ice for pre-cooling.
6 Instruments and Equipment
6.1 Constant-temperature water bath kettle: the temperature can reach 37 °C ± 1°C.
6.2 pH meter, with an accuracy of 0.1.
6.3 Electronic balance, with an accuracy of 0.01 mg.
6.4 High-speed centrifuge.
6.5 Flow cytometer.
6.6 Constant-temperature oscillating shaker: the temperature can reach 37 °C ± 1°C;
the speed range satisfies 125 r/min ~ 250 r/min.
6.7 Ultraviolet - visible light spectrophotometer: the detectable wavelength includes
600 nm ± 20 nm; equipped with a 1 cm cuvette.
7 Samples
7.1 Test Microorganisms
Salmonella typhimurium NM2009, including plasmid expressing umuC-lacZ gene and
ampicillin resistance gene (see Appendix A for the plasmid sequence).
Then, quickly add 500 μL of PI solution to the above-mentioned 1.5 mL centrifuge tube;
place the mixture on ice to react for 1 h. Before performing flow cytometry, it shall be
kept on ice.
8.4 Determination by Flow Cytometer
Use the flow cytometer to perform flow cytometry on the dyed sample. Set the
excitation wavelength to 488 nm. For the fluorescence intensity measurement of FDG
hydrolysate fluorescein, the reception wavelength is 525 nm (FL-1 channel); for PI
dyeing florescence measurement, the reception wavelength is 670 nm (FL-2 channel).
Set the total number of cells to be measured to 5,000 ~ 10,000.
8.5 Threshold Setting
Through the forward angle scattered light FSC and the side angle scattered light SSC,
circle the target bacteria, so as to remove the influence of multi-cell adhesion on the
result.
Through separate PI dyeing, determine the relative florescence values of the FL-1
channel and the FL-2 channel. On the FL-1 coordinate axis, circle the PI-dyed cells.
The relative florescence value of the FL-1 channel of the cells in the circle has a range,
and the cells with the FL-1 relative fluorescence value not less than this range are PI-
dyed negative cells.
Through separate FDG dyeing, determine the relative florescence values of the FL-1
channel and the FL-2 channel. On the FL-2 coordinate axis, circle the FDG-dyed cells.
The relative florescence value of the FL-2 channel of the cells in the circle has a range,
and the cells with the FL-2 relative florescence value in this range are FDG-dyed
positive cells.
For samples simultaneously dyed with FDG and PI, select the FDG dyed positive cells
and PI-dyed negative cells as the target cell population for the subsequent data
processing and result analysis.
9 Test Data Processing
SOS induction coefficient shall be calculated in accordance with Formula (1):
Where,
Fi---SOS induction coefficient;
Am---the average relative fluorescence value of FDG hydrolysate fluorescein of
samples treated with mutagenesis source;
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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