BACKGROUND
Chemical mutagenesis is a technique for inserting mutations into DNA by utilizing chemicals that damage or modify the nucleotides. By introducing random or site-directed mutations, chemical mutagenesis can be employed to investigate gene function, protein structure, or regulatory elements.
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Mutagenesis can be accomplished using a variety of substances, including nitrosoguanidine, ethyl methanesulfonate, hydroxylamine, and bisulfite. Each chemical has a unique method of action as well as a mutational spectrum. Nitrosoguanidine, for example, produces transitions and transversions by deaminating cytosine and adenine, whereas ethyl methanesulfonate produces transitions by alkylating guanine.
Depending on the organism, the chemical, and the desired mutation rate, the technique for chemical mutagenesis may differ. A general protocol, on the other hand, consists of three steps: presoaking, mutagen application, and washing. Presoaking hydrates the cells while increasing their permeability to the mutagen. Mutagen application is used to expose cells to a chemical for a set amount of time. Washing is performed to remove excess mutagen and avoid additional damage.
The concentration and duration of the mutagen, the temperature and pH of the solution, the type and stage of the cells, and the presence of DNA repair mechanisms are all parameters that can affect the efficacy and specificity of chemical mutagenesis. As a result, optimizing these parameters for each experiment and applying proper controls to evaluate the frequency and spectrum of mutations is critical.
Chemical mutagenesis is an effective method for creating genetic diversity and researching biological functions. It does, however, have significant limitations, like being generic, random, and potentially hazardous. As a result, it should be handled with caution and in accordance with all safety rules.
Mutagenesis of existing strains to create overproducing mutants is one way. Chemical mutagenesis has been widely employed in bacteria, particularly lactic acid bacteria, and is considered food-grade because it does not include the introduction of heterologous DNA or recombinant DNA manipulation. The possibility of a single exposure to two chemical mutagens for the isolation of -gal overproducing mutants of Bifidobacterium spp., Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were investigated in this study.
REQUIREMENTS
- Bacterial strains (Bacillus, Lactobacillus, Streptococcus sp.)
- BHI (Brain heart infusion) broth
- Ethyl methanesulfonate (EMS)
- N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)
- X-gal agar medium
- β-galactosidase
- Rifampicin
- Petri plate
- Glass spreader
- Test tubes
PROCEDURE
- Select 2-3 types of bacterial strains (Bacillus, Lactobacillus, Streptococcus sp.) [1]
- All bacterial strains are inoculated in 2 sets of 5 ml of BHI broth medium (Brain heart infusion) and incubated anaerobically at 37C for 48 hours.
- Small amount of ethyl methanesulfonate (EMS) is added to one set and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) is added to another set. [1]
- To screen mutagen, X-gal medium containing B-gal. rifampicin as an antibiotic.
- Spread the culture on the X-gal medium plates, after a specific interval of time (0, 30, 60, 120 sec., etc) and incubate overnight. [2]
- Observe for the blue color colonies on a plate, those are selected as mutated colonies
- Plot survival curve time V/S colony.
CONCLUSION
The procedure outlines a method for inducing mutation in bacterial strains (Bacillus, Lactobacillus, Streptococcus sp.) using chemical mutagens EMS and MNNG. The mutagenized bacterial strains are then screened using X-gal medium plates containing B-gal and rifampicin to select for mutated colonies.
The survival curve time V/S colony plot helps evaluate the mutagen’s impact on bacterial survival rate and colony formation. The appearance of blue-coloured colonies on the X-gal plates indicates the occurrence of mutation.
REFERENCES
- S. A. Ibrahim, and D. J. O’Sullivan, “Use of Chemical Mutagenesis for the Isolation of Food Grade β-Galactosidase Overproducing Mutants of Bifidobacteria, Lactobacilli and Streptococcus thermophilus”, Journal of Dairy Science Vol. 83, No. 5, 2000.
- Agrawal, S., S. K. Garg, and S. M. Dutta. 1989. Microbial β-gal: Production, properties and industrial applications. A review. Indian J. Dairy Sci. 42:251–262
- Takayasu Ito, Takanori Nakamura, Hisaji Maki, Mutsuo Sekiguchi, “Roles of transcription and repair in alkylation mutagenesis”, Mutation Research/DNA Repair, Volume 314, Issue 3, May 1994, Pages 273-285.
- Todd H. Dechter and Dallas G. Hoover, “Survivability and β‐galactosidase activity of bifidobacteria stored at low temperatures”, Food Biotechnology, Vol. 12, 1998.
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FAQs
Chemical mutagenesis is the process of inducing mutations in DNA using chemical agents, such as EMS and MNNG. These agents can modify the DNA sequence by altering or damaging the bases, leading to mutations.
Chemical mutagenesis is useful in genetic research because it allows for the creation of specific mutations in DNA sequences. This can be used to study the function of specific genes or to develop new strains of organisms with desirable traits.
Chemical mutagenesis involves the use of potentially hazardous chemicals, which can pose risks to both researchers and the environment. Safety precautions should be taken when handling these chemicals, including the use of personal protective equipment and proper disposal methods.
