Mutagenic effect of nitrosoguanidine and screening experiments with nutrient-deficient strains of bacteria
Mutagenic effect of nitrosoguanidine and screening experiments with nutrient-deficient strains of bacteria
Wild-type strains obtained from nature are generally able to grow on basic medium. After physical or chemical treatment, some of the wild-type cells have mutated genes, losing the ability to synthesize certain essential substances (such as amino acids, vitamins, bases, etc.), and thus cannot grow in the basic medium, and can grow only when the basic medium is supplemented with such substances. This kind of mutant strains showing some kind of defects in nutrition is called nutrition-deficient strains, which is an indispensable and important material in genetics, molecular biology research and practical application. Source: Laboratory Course in Genetics
Operation method
basic program
Principle
Nitrosoguanidine (NG, NTG) is a widely used strong mutagen. It induces a GC→AT transition mainly in the replication region of the DNA strand, and it can produce high-frequency mutations in microorganisms even under conditions of low lethality. In this experiment, after the mutation of Corynebacterium glutamicum by using nitrosoguanidine, the nutrient-deficient type was detected by the point-species method, and then characterized by the growth spectroscopy method to obtain the nutrient-deficient strains and to determine the specific substances of the nutrient defects.
Materials and Instruments
Strain : Corynebacterium glutam icum T-13:bio - CB solid medium CB slant MM slant Citrate buffer(pH5.5) Phosphate buffer(pH7.0) Physiological Saline Move (I) Mutagenic treatment 1. Preparation of bacterial suspension Pick a few slant strains into 5ml CB culture medium, set at 30℃ and incubate for 8h with shaking (200r/min), take 0.5ml of culture into another 5ml CB culture medium and continue to incubate overnight with shaking. The overnight culture was centrifuged at 4000 r/min for 8 min, and the supernatant was discarded; 1 ml of citrate buffer (pH 5.5) was added into the centrifuge tube to stir the precipitate, and then 4 ml of the same buffer was added to make the bacterial suspension; the bacterial suspension was washed by centrifugation at 4000 r/min for 8 min for two times, and then 5 ml of the buffer mentioned above was added to make the bacterial suspension (about 108 organisms/ml). Finally, 5 ml of the above buffer was added to make a bacterial suspension (about 108/ml). 2.Nitrosoguanidine mutagenic treatment Take two small sterile test tubes and add 2 ml of bacterial suspension respectively, preheat in 37℃ water bath. One of them carefully added 50μl NTG solution (original concentration of 2mg/ml), mixed well, the two test tubes at the same time in a 37 ℃ water bath insulation for 30min. remove the two insulated test tubes by centrifugation at 4000r/min for 8min, carefully discard the supernatant (supernatant placed in the vessel containing 1mol/L NaOH solution, do not drop the supernatant containing NTG on the skin or clothing). Do not drop the NTG-containing supernatant onto the skin or clothes), add 5 ml of phosphate buffer (pH 7.0) to make the bacterial suspension, centrifuged for 8 min, and washed repeatedly for two times, and finally added 2 ml of phosphate buffer to make the bacterial suspension. Take 2 kinds of bacterial suspension 0.5ml each according to the 10-fold dilution method to dilute the bacterial suspension to 10-6, respectively, take 10-4, 10-5 and 10-6 dilution spread on the CB plate, each dilution coated with 9 dishes; similarly, take the bacteria without mutation treatment dilution of the bacteria diluted in each dilution coated with 3 dishes as a control; placed in 30 ℃ incubation for 2 d. Statistics on the number of CFU on the plate to calculate the bactericidal rate. The colonies on the plate will be used to detect the colonies. (ii) Detection of nutritional defects 1. Spot seeding method Take the basic medium (MM) and complete medium (CB) plate, with a marker at the bottom of the plate to make the corresponding mark (i.e., a set of MM plate and a set of CB plate corresponds to). Place the pre-numbered grid paper (see Figure 23-1) on the bottom of the corresponding plate. Carefully dip a sterile toothpick into the surface of the colony to be tested and place it on the corresponding position of the MM and CB plates respectively (MM plates first, followed by CB plates. Select as many colonies as possible), and incubate at 30℃ for 2d. 2. Photocopying method Take 1 dish each of MM and CB plate, and draw an arrow at the bottom of the plate as a direction mark. Select the plate with dispersed colonies as the mother plate, mark the same direction, and mark the direction on the sterile photocopy plate. Place the mother plate upside down on the flannel of the photocopier plate according to the marked direction, then remove the photocopier plate, place the MM plate upside down on the photocopier plate according to the same method, remove the MM plate, and print the cells on the photocopier plate onto the CB plate in the same way. Pairs of photocopied plates were incubated at 30℃ for 2d. 3. Sandwich method Take sterile petri dishes 3 dishes, each pour a thin layer (about 5 ml) of MM medium, after solidification, add 0.1 ml of 10-5 diluted bacterial mutation treatment, and then pour about 5 ml of MM medium (melting a little cold, not scalding can be, the temperature of about 45 ℃), shaking well, to be solidified plus a layer of about 5 ml of MM medium, after solidification, placed in 30 ℃ culture for 2 d. At the bottom of the plate above will be growing At the bottom of the above plate, mark the growing colonies, count the number of colonies, add a layer of about 5 ml of semi-solid CB medium, and incubate at 30 ℃ for 2d. Among the three methods mentioned above, the colonies that could not grow on the MM medium but grew in the corresponding position on the CB plate needed to be further verified to see whether they were truly nutrient-deficient or not. These may be defective colonies number, respectively, corresponding to the inoculum in the MM slant and CB slant, placed in 30 ℃ culture for 2 d. In the MM slant does not grow, but in the CB slant on the growth of strains saved for further identification. (C) the identification of nutritional deficiencies 1. Preliminary determination Gently pick a ring of the strain to be tested in 0.5 ml of saline, shaking to make a bacterial suspension. Take 0.1ml of the suspension and spread it evenly on the MM plate. After drying, the bottom of the plate was divided into three areas according to Figure 23-2, and labeled A, V and B. The nutrient mixture was gently placed in the center of the corresponding area with a sterile toothpick (but not too biased towards the center or edge of the plate), and incubated at 30℃ for 2d to determine the nutrient defects of the three areas according to the growth of the three types of nutritional deficiencies. 2. Accurate identification According to the above method, the preliminary determination of the defective strains were made into bacterial suspension and spread on the MM plate. Divide the area at the bottom of the plate (the number of divided areas is the same as the number of groups of growth factors to be added). Add a small amount of nutrient mixture according to the area, incubate at 30℃ for 2d and observe the results. Caveat 1. Put all the instruments that have been in contact with nitrosoguanidine in a ventilated place and soak them in 1 mol/L NaOH solution to decompose and destroy the residual nitrosoguanidine, and then wash them with water. 2. When photocopying and identifying defective strains, the growth on MM and CB medium should be observed in time to avoid affecting the accuracy of the results. 3. Preliminary identification and accurate identification of bacterial liquid cell density can be slightly higher, so that the bacteria to be tested evenly distributed on the surface of the culture medium. For more product details, please visit Aladdin Scientific website.
CB culture medium CB solid medium CB slant MM medium MM slant Citric acid buffer (pH 5.5) Phosphoric acid buffer (pH 7.0) Physiological saline Nitrosoguanidine Mixed amino acids Mixed vitamins (mixed with 10 equal amounts of vitamins) Alkali mixture (mixed with 6 equal amounts of bases).
Constant temperature incubator Oscillator Triangular flask Test tubes Straws Photocopying plates Toothpicks
