Isolation of antibiotic resistant Spontaneous mutant

BACKGROUND

A heritable alteration to the DNA’s nucleotide sequence is known as a mutation. Depending on the type of genotypic alteration that has taken place or the effects it has on phenotype, mutations can be classified. A mutation may change a microorganism’s phenotypic in a variety of ways. The colony or cellular morphology of the microorganism is altered through morphological alterations.

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A gene that encodes an enzyme engaged in a metabolic pathway of amino acid synthesis may experience nutritional or biochemical change. When a gene encoding a transcription factor is altered, changes in gene regulation follow. Lethal mutations stop an organism from being able to reproduce, and when they manifest, they cause the microorganism to perish.

A microbe’s metabolic pathway is regularly rendered inactive by mutations, and this frequently prevents the microorganism from growing on media that are deficient in the process’s end product. Microorganisms are categorized as prototrophic and auxotrophic based on this theory. The dietary needs of prototrophic organisms (of the wild type) are identical to those of their predecessors.

For survival and growth, they only require inorganic salts, an organic energy source like sugar, fat, or protein, and water. In other words, “Minimal media” is all that the prototrophs require for development and survival. Mutants that are auxotrophic are unable to grow in the absence of one or more crucial nutrients. For specific enzymes in the nutrition production pathway, auxotrophs are mutant.

Whether it happens in a bacteria or a eukaryote, such an error is referred to as an inborn error of metabolism. Only enriched media that supply the specific nutrient that the mutant cannot metabolize on its own can support the growth of auxotrophs.

Types of mutations:

Spontaneous mutations and induced mutations are the two types of mutations.

Spontaneous mutations are mutations for which there is no established cause. Due to the low frequency of metabolic errors or mistakes made during DNA replication, this leads to the chemical instability of purine and pyrimidine bases.

Mutations brought on by organisms being exposed to mutagenic substances like ionizing radiation, UV light, or other compounds that interact with nucleic acids. These mutagens are frequently used by researchers to boost the mutation rate in experimental organisms.

In general, chemical mutagens cause little chromosomal defects called point mutations, whereas ionizing radiation causes massive chromosomal abnormalities. Simple base-pair modifications, base-pair substitutions, base-pair duplications, and base-pair deletions are all examples of point mutations.

A single location on a chromosome is the site of a point mutation. A nonsense mutation is a type of point mutation that converts a normal codon into a stop codon that does not code for an amino acid and arrests peptide synthesis without amino acid insertion, resulting in a protein product that is not functional. Missense mutations are point mutations in which a single nucleotide is changed to result in the substitution of a different amino acid.

Antibiotic resistant Spontaneous mutant

The addition or deletion of nucleotides that are not multiples of three results in frame shift mutations, which cause the codon to be read erroneously during translation. A silent mutation has no impact because it only results in base substitution and not amino acid substitution. Those substitutions won’t affect the product in any way and can’t be found without genome sequencing.

The mutation events are frequently reversible in any case. The successive nucleotide pair mutations bring back the original wild type phenotype. In other words, a gene that has experienced a mutation returns to its initial base structure. Reversion, reverse mutation, or back-mutation are terms used to describe this.

In bacteriology, isolation of mutant organisms is frequently the first step in genetic and biochemical investigations. Since they develop in the presence of antibiotic concentrations that prevent the growth of normal bacteria, the spontaneous mutations brought on by resistance to antibiotics like streptomycin are simple to spot.

GRADIENT PLATE TECHNIQUE

REQUIREMENTS

• 24 hr old grown Escherichia coli culture
• Nutrient broth
• Nutrient agar tubes
• 90% ethanol
• Pipette
• Glass rod spreader
• Water bath

PROCEDURE

1. Gradient plate preparation ^[1]
   1. Prepare the nutrient agar and autoclave it.
   2. Pour the nutrient agar into a sterile petri plate. By putting either a glass rod under one side, the medium can solidify in a slanting posture.
   3. Remove the glass rod after the agar media has settled and set the plate in a horizontal position.
   4. Fill the second tube of the second nutritional agar medium with 0.1 mL of 1% Streptomycin solution using a pipette.
   5. Pour the contents into the tube while rotating it between your palms, covering the gradient layer of agar. Afterwards, place the medium on a flat surface and let it set.
   6. On the plate's bottom, indicate where the antibiotic concentration is low and high.
2. Inoculation of culture ^[2]
   1. Pipette out 200 µL of the overnight grown E. coli culture.
   2. By rotating the plate, a sterile bent glass rod can be used to evenly distribute the inoculums around the agar surface.
   3. For 48–72 hours, incubate the infected plate inverted at 37°C.
   4. Keep track of the results as you watch the plate for the development of E. coli colonies in the low- and high-streptomycin areas.
3. confirming the presence of E. coli colonies that are resistant to streptomycin
   1. Choose and mark an isolated E. coli colony on the Nutrient agar plate in the HSC area.
   2. Using a clean inoculating loop, choose the chosen colony and streak it in the direction of the HSC region on a second gradient plate.
   3. Repeat this process using one or two colonies of mutants from the HSC area that are resistant to streptomycin.
   4. The inoculation plates should be incubated at 37°C for 24-72 hours in an inverted position.
   5. Take note of the expansion of streaking colonies near the HSC area.

CONCLUSION

As a result of the above procedure, colonies that arise in the region of high concentrations of streptomycin (HSC) will be mutants that are resistant to streptomycin, and the growth of E. coli colonies in HSC shows the isolation of these mutants has been effective.

REPLICA PLATE TECHNIQUE

REQUIREMENTS

• 24 hr old grown Escherichia coli culture
• Nutrient broth
• Minimal salt agar with glucose
• Nutrient agar
• Streptomycin solution (10mg Streptomycin, 100ml of sterile distilled water)
• Petri plate
• wooden replica block
• velvet cloth
• ethanol
• glass rod
• colony counter

PROCEDURE

1. Prepare the nutrient agar and autoclave it.
2. pour the molten agar into the petri plate and allow it to solidify it.
3. In the third tube of molten nutritional agar (kept at 55°C), add 0.1% of streptomycin using a sterile pipette. After thoroughly mixing between your hands, pour the contents into a sterile petri dish. To be allowed to set. ^[1]
4. On the surface of the nutrient agar plate, spread 200 µL of the E. coli test culture.
5. Spread the inoculum uniformly on the plate using a bent glass rod that has been flamed and soaked in alcohol.
6. At 37°C, incubate the plate inverted for 24-48 hours.
7. See the E. coli colonies on the plate after incubation; this plate was chosen to serve as the master plate.
8. The bottom of the master plate, nutrition agar plate, and streptomycin-supplemented plate all included a reference mark.
9. The E. coli colonies on the master plate were gently pressed onto by the sterile velveteen colony carrier.
10. The sterile velvet cloth was gently placed on the nutrient agar plate, then the nutrient agar plate with streptomycin added, without moving the carrier.
11. For 48–72 hours at 37°C, incubate the infected plates, nutrition agar, and streptomycin agar plates inverted. The master plate is refrigerated.
12. Using a Colony Counter, the number of colonies in replica plates containing nutrient agar and streptomycin agar were counted after incubation.
13. We observed and compared the colonies that appeared on the Streptomycin and Nutrient Agar plates.

CONCLUSION

The Colony Counter was used to count the number of E. coli colonies that appeared on the streptomycin-supplemented agar and were confirmed to be mutants that are resistant to streptomycin.

REFERENCES

1. https://vlab.amrita.edu/?sub=3&brch=76&sim=1089&cnt=1
2. Herve Nicoloff,Vincent Perreten, and Stuart B. Levy, “Increased Genome Instability in Escherichia coli lon Mutants: Relation to Emergence of Multiple-Antibiotic-Resistant (Mar) Mutants Caused by Insertion Sequence Elements and Large Tandem Genomic Amplifications”, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol. 51 (4), April 2007.
3. Sophia Silvia, Samantha A. Donahue, Erin E. Killeavy, Gerwald Jogl and Steven T. Gregory, “A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus”, Antibiotics journal, 2021.
4. ABRAHAM L. SONENSHEIN, BRIGITTE CAMI, JEAN BREVET, AND RICHARD COTE, “Isolation and Characterization of Rifampin-Resistant and Streptolydigin-Resistant Mutants of Bacillus subtilis with Altered Sporulation Properties”, JOURNAL OF BACrERIOLOGY, Vol. 120 (1), October 1974.

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• Determination of Thermal Death Point of bacteria
• Microbiological assay of antibiotics using cup plate method

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