Selection and characterization of mutant cell lines, auxotrophic mutants
Isolation of Mutants:-
Introduction:-
> Mutation occurring in microorganism can be detected and efficiently isolated from the parent organism of other mutants.
> While studying we must be aware of wild type characters of an organism ,so the mutants can easily detected.
> In bacteria and other haploid microorganism, the detection system are straight forward because any new allele should be observed immediately.
> In albino mutation, the detection is very simple. It requires only change in colour of bacterial colony. > The other detection systems are rather complex.
Some Detection Methods:-
1.Replica plating technique
2. Resistance selection method
3. Substrate utilization method
4. Ames method
1.Replica plating technique:-
> Joshua and Esther Ledgerberg (1952) developed a new technique called replica plating .
> This technique is used to detect auxotrophic mutants and wild type strains on the basis of ability to grow in the absence of amino acids.
> Also this test is used to demonstrate the presence of antibiotic resistance in bacterial cultures prior to exposure of antibiotic
> Steps Involved:-
i. Generate the mutants by treating a culture with a mutagen e.g.nitrosoguanidine .
ii. Inoculate a plate containing complete growth medium and incubate it at proper temperature. Both wild type and mutant survivors will from complete medium.
iii. This plate containing complete medium is called master plate.
iv. Prepare a piece of sterile velvet and gently on the upper surface of the master plate to pick up bacterial cell from each colony.
v. As pressed the master plate, again gently press the velvet on the replica plates containing complete medium in one set and lacking cine in only leucine in the other set.
vi. Thus, the bacterial cells are transferred in replica plates in the same position as in master plate.
vii. Incubate the plates and compare the replica plate with master plate for bacterial colony not growing on replica plate.
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2. Resistance selection method:-
> This is another method used for isolation of mutants.
> Generally the wild the wild type cells not resistant either to antibiotics or bacteriophage.
> Therefore, it is possible to grow the bacterium in the presence of agent.
> This method is applied for isolation of mutants resistant to chemical compounds that can be amended in agar, phage resistant mutants.
3. Substrate utilization method:-
> This method is employed in the selection of bacteria. Several bacteria utilize only a few carbon sources.
> The cultures are plated on to medium containing alternate carbon sources.
> Any colony that grows on medium can use the substrate and are possibly mutants. These can be isolated.
> Sugar utilization mutants are also isolated by means of color indicator plates.
> EMB medium is used for this purpose.
> This medium contain lactose sugar as carbon source and complete mixture of amino acids.
> Therefore both lactose wild type and lactose mutant cells can grow and form colonies on EMB agar plates.
> The lac+ cells catabolize lactose and secrete acids,therefore the pH of the medium decreases. This will result in staining of colony to dark purple.
> On the other hand, Lac- cells are unable to utilize lactose and use some of the amino acids as carbon source.
> After utilization of amino acid, ammonia is produced that increases the pH and de colorize the dye resulting in white colony.
4. Ames method:-
> Ames test In 1974 Bruce Ames developed a method for evaluating the potential of chemical to cause cancer, known as Ames test .
> Ames test is based on the principle that both cancer and mutations results from the damage of DNA, and results of experiments have demonstrated that 90% of known carcinogen are also mutagens.
> Several species of salmonella typhimurium are employed. Each strain contains a different mutation in the operon histidine biosynthesis.
> Steps:-
i. Prepare the culture of Salmonella histidine auxotrophs (His-).
ii. Mix the bacterial cells and test substance( mutagen) in dilute molten top agar with a small amount of histidine in one set, and control with cmplete medium plus large amount of histidine.
iii. Pour the molten mix on the top of minimal agar plates and incubate at 37°C for 2-3 days.
iv. Until histidine is depleted all the His- cells will grow in the presence of test mutagen.
v. When the histidine is completely exhausted only the revertants will grow on the plate.
vi. The number of spontaneous revertants is low, whereas the number of revertant induced by carcinogen is quite high.
vii. High number of colonies represent the greater mutagenicity.
viii. A mammalian liver extract is added to the above molten top agar before plating.
ix. The extract converts the carcinogen in to electrophilic derivatives which will soon react with DNA molecule.
x. In natural way it is occurs in mammalian system when foreign particle are metabolized in the liver.
xi. Bacteria does not have metabolizing capacity, therefore, the liver extract is added to this test, to promote transformation.
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Auxotrophic mutants:-
> These are bacteria, yeast, protoplast or mammalian host cell strains that can’t produce a nutrient vital for growth due to genetic mutations. As a result, these strains are unable to survive in media lacking that specific nutrient unless it is provided externally.
> The term auxotrophy refers to a nutritional dependency, where the mutant requires an auxiliary or supplemental source of the missing nutrient for its growth and survival. In contrast, the corresponding wild type strain can synthesize that specific nutrient and is not dependent on an exogeneous supply in the growth medium.
> Auxotrophic mutants are often used in genetic studies, selection experiments, and research involving metabolic pathways and nutrient utilization. They are also very useful in gene cloning procedures.