GB/T 40186-2021Determination of genetic material damage strength for microbial mutation breeding—Umu method (English PDF)

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.

5.4 Fluorescein di-beta-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.

7.1 Test Microorganisms

Salmonella typhimurium NM2009, including plasmid expressing umuC'-'lacZ gene and ampicillin resistance gene (see Appendix A for the plasmid sequence). […]

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.