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Frame Shift Mutation: The polypeptide chain codes for an entirely different protein if there is a change in the nucleotide. These mutations are caused when chemical binds to DNA, so chemicals are known as intercalating agents. In the distorted region replication occurs and this results in the deletion or insertion of the base pairs and is called frameshift mutation if not multiple of three.
Silent Mutation: In the silent mutation there won't be any change in the amino acid sequence of the protein (6), can seen in non-coding region or within an exon and cannot make any difference in final amino acid
Deletion Mutation: In deletion mutation, a part of the sequence or chromosome of DNA is missing.
Insertion Mutation: Insertion mutation is the mutation in which there will be addition of one or more nucleotide base pairs in to a sequence of DNA.
Bases can differ from few to thousands, deletion or insertion of two or more bases or multiple of three which can results in frameshift mutation or shift in the reading frame.
Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will include errors commonly. In order to do this the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental).
Function of mutS: In an ATP pathway, during DNA replication the included mismatch nucleotide is identified by MutS protein. The mechanism carried by this kind of protein is that they interact with a mismatch pairs and then there will be formation of hydrogen bond between glutamate residues.
Strand specificity is known as mismatch repair system. New strand obtained from template strand contains errors, so the mismatch repair system works to differentiate the newly synthesized strand that is called daughter strand from the parental or template strand.
Consequences for a muts: If muts is non-functional then the recombination results in similar sequences which leads to an increase in the mutation rate and can be deleted when mutS is functional (6). Increase in recombination rates takes place if there is a deficient mutants and determination is carried out by transformation with DNA incorporation. DNA mismatch is binded by the MutS, mutS of DNA complex is joined by MutL and it activates MutH.
Description of drugs:
Rifampicin: It is a semi synthetic compound derived from Amycolatopsis rifamycinica(2). It belongs to rifampicin group. It eliminates bacteria that cause tuberculosis. It works best on a empty stomach one hour before meal or at least two hours after a meal (3).
Chemical structure of Rifampicin(2):
Drug action and target: The action of rifampicin includes the binding of β-subunit by inhibiting the dependent RNA polymerase, thereby prevents the translation of proteins and transcription to RNA. It is a good drug for lipophilic action and also used to treat tuberculosis of meningitis (2).
Streptomycin: Streptomycin is a bactericidal (kills bacteria) antibiotic. It is derived from the actinobacterium Streptomyces griseus and belongs to aminoglycosides class. It cannot be given orally but must be administered by regular intramuscular injections. The adverse effect of streptomycin medicine is ototoxicity, which can cause ear damage (4).
Chemical structure of Streptomycin(4):
Drug action and target: Streptomycin inhibits protein synthesis and binds to 30S ribosomal subunit of S12 protein, the 30S ribosomal subunit interfere with formyl-methionyl-tRNA binding and this can keep from occurring to the synthesis of protein and latter leads to cell death or lysis. If the drug level of concentration is low then it can be able to inhibit the bacterial growth to lead the misreading of mRNA by prokaryotic ribosome. Gram negative and Gram positive bacteria are inhibited by streptomycin drug so it is most commonly used as the antibiotic class of broad spectrum (4).
Nalidixin: It is the first of the synthetic quinolone antibiotics. This drug is effective against Gram- negative and Gram-positive bacteria. It has a bacteriostatic effect only if the drug is given in low concentration and thereby which can prohibit the growth or inhibit. For bactericidal, a high concentration is used to kill the bacteria. The drug is commonly used in urinary tract infection treatment which is caused by different microorganisms such as E. coli, Shigella, etc.
Chemical structure of Nalidixin(5):
Drug action and target: The DNA replication in bacteria is blocked by the naladixin and DNA gyrase subunit inhibition takes place by complex analogue which can be induced. It has the capability to inhibit DNA gyrase subunit by nicking, thereby which produces a supercoiled DNA by positive stress binding (5).
Polymerase chain reaction sequencing: It was developed in 1983 by Kary Mullis and is used for the genotypic analysis and identification of the organisms. One colony is taken and suspended in the 50µl of water. 1µl of the DNA preparation is taken into the ready-to-bead which is used for PCR (it contains DNA polymerase, dNTP's, buffer, salts) and into that 1µl of primer 1, 2 are added according to the antibiotics used finally water is added so that the volume in the tube should be of 25µl
PCR reaction: Initially denaturation for 5mins at 95°C, 30 cycles and each will contain 90 s at 95°C, 50°C and 150 s at 72°C and followed by a extension for 7minutes at 72°C with the PCR products the gel electrophoresis is carried out by taking 1% agarose gel which contains EtBr/100ml gel solution latter 1kb of DNA ladder is used this whole setup is carried out for 1hour at 150V.
Sanger DNA sequencing: Sanger DNA sequence was first introduced by Fred sanger and Alan coulson in 1975 which is an direct sequencing of DNA method and is called as plus-minus method. Firstly the synthetic radio-labelled oligonucleotides are primed with DNA polymerase after that it generates DNA fragments and finally analysis of fragments is carried by auto-radiography and electrophoresis. This kind of sequencing is used to detect 5386kp of bacteriophage É¸X174 genome.
Dideoxynucleotides are also known as ddNTP's, on deoxyribose sugar of ddNYP's does not have 3'-hydroxyl group. These nucleotides are very helpful in DNA sequencing.
In electrophoresis, the dideoxynucleotides plays a role in the DNA sequencing and latter allowed running PCR. This PCR contains different combination or mixture of nucleotides those are, four of them are deoxynucleotides and one is dideoxynucleotide. Presence of dideoxynucleotides complements the length of the strand for equal bases. After electrophoresis of the sample, each band length complement will represents the presence of dideoxynucleotides.
Strand termination is a transcriptional termination proceeds when there is an elongation factor or reaction that which encounters with a core RNA polymerase. So, by this a DNA template and an enzyme release RNA.
Materials and Methods:
Overnight culture of wild and mutant strains of E.coli MG1655
Flasks, Pipettes, tubes, centrifuge, PCR machine, DNA sequence, gel tray, power supply, pure Taq ready to go PCR beads, primers, QIAGEN PCR purification kit
Agarose, 1XTAE buffer, EtBr, 0.9%NaCl, nalidixin, LB medium, LA plates, Mueller-Hinton plates, LA plates with nalidixin, DNA ladder.
Solid media: LA (Luria Agar) plates were used to carry out the whole experiment. The media contains casein enzymatic hydrolase, yeast extract, sodium chloride and agar. Casein enzymatic hydrolase and yeast extract act as growth factors by providing nitrogen, carbon, mineral sources. Sodium chloride maintains osmotic balance. This is a nutrition rich media with simple composition is mostly used for the cultivation and growth of E.coli.
Muller Hinton Agar: The components of agar are beef extract, acid hydrolysate of casein, starch and agar. Beef extract and casein gives all nutrients such as vitamins, minerals required for the growth of the microbe, the toxic products produced by the microbes are taken up by starch. Thus this agar is mainly used for antimicrobial susceptibility test.
E-Test: Epsilometer Test (E-Test) is a method used to find how effective the microbe takes up the specific antibody to which it is exposed. It works on the principle of antibiotic diffusion in the agar. A strip containing different concentrations of antibiotic creates an antibiotic gradient. When this strip is placed on the agar the antibiotic in the strip starts to diffuse and creates a zone of inhibition. The end point where the zone meets gives us the minimal inhibitory concentration value, this method is simple (7).
PCR: One colony from wild and mutant plates was taken in a loop and suspended in 50µl of water and was boiled for 5 minutes. After the boiling is complete, to 1µl of the DNA in ready to go PCR beads 1µl forward primer nal 1(5'-GAGGAAGAGCTGAAGAGCTCCT-3') and reverse prime nal 2 (5'-CCGGTACGGTAAGCTTCTTCAA-3') was added. This sample was run in PCR at following conditions initial denaturation 95°C, 5min and 35 cycles each of 94°Cfor 30seconds, 55°C for 30 sec and 72°C for 1min and final extension at 72°C for 7min. PCR products were examined by electrophoresis on 1% agarose gel containing 1 μl EtBr per 100 ml gel solution for staining the gel. The electrophoresis was for 1 hour at 150 V. DNA ladder was as a marker used to compare the size of the DNA band obtained.
The PCR product is then purified using QIAquick PCR Purification kit and analysed using BLAST search database.
0.2 ml of wild type (wt) and âˆ†mutS strains of E.coli were taken from 3ml of overnight culture. It was spread on LA plates containing the antibiotic nalidixin. Another 0.2 ml of the culture was taken and serially diluted. 103 and 102 dilutions were selected and were spread on LA plates without antibiotic. All the plates were incubated at 37C for overnight.
On day 2, the mutation frequency was calculated counting the colonies formed in wt and âˆ†mutS on the LA plate with the antibiotic. The mutation frequency was calculated by dividing the colony forming unit of the antibiotic plate by CFU of the same culture without antibiotics. Thus the value obtained was 2.5* 10-8 for wt which was lower than âˆ† mutS strain which gave a value of 9.4 * 10 -6 which indicates the mutation level is higher for mutant strains than the wild type. One to three colonies from LA plates containing nalidixin was restreaked on to a new plate.
One colony from each strain was picked from LA plate without nalidixin and resuspended in 1ml of 0.9% NaCl and vortexed. Then the suspension is spread on Mueller-Hinton plate using sterile swab. After the plate dries Etest strip is placed and the plate was incubated at 37C for over- night.
On day 3, the Minimal Inhibitory Concentration was calculated for the plates containing the Estrips.
On the fourth day of the experiment colony PCR was carried out for the re-stroked mutants. The PCR product was then run on agarose gel to confirm the presence of DNA and then went for sequencing the sample.
The MIC value was calculated was 4µg/ml for both wild type and âˆ†mutS strains. The mutation frequency for wild type and âˆ†mutS was calculated as 2.5*10-8 and 9.4*10-6. The average difference in mutation frequency (mutS/wt) calculated was 6.03*102.
From the BLAST search mutations were identified and since we had so many mutations in our sequence. We analyzed the sequence of other group (group: 12) and found that both wild and âˆ†mutS strains had mutations.
Mutation at nucleotide level: Point mutation occurred in both wild and âˆ†mutS strain, A245G and C248T respectively.
Mutation at Amino Acid level: Wild type showed a point mutation Asp82GGC and no mutation occurred in âˆ†mutS, Ser82TCG.
For both the strains wt and âˆ†mutS intersection of the clear zone with the Estrip was at 4µg/ml which depends upon the mutation frequency. In the case of wt the mutation frequency calculated is 0.25*10-7 which gives the probability of the presence of 1 or less mutants and 97*10-7 was the mutation frequency calculated for âˆ†mutS where the presence of mutants ranges from 97-100 which is too low. When comparing the value there is no significant difference between the two strains and hence the MIC value is the same.
From the BLAST search mutations were identified and since we had so many mutations we analysed the sequence of other group (group: 12) and found that both wild and âˆ†mutS strains had mutations.
Mutation at nucleotide level: Point mutation occurred in both wild and âˆ†mutS strain the mutations were A245G and C248T respectively. Where G was replaced by A at 245th position and T was replaced by C at 248th position. Thus the mutation occurs between purine and pyrimidine. This is called a transition.
Mutation at Amino Acid level: In the case of wild type mutation was Asp82GGC and in âˆ†mutS it was Ser82TCG. In the case of wild type strain, Asp was replaced by GGC codon. Thus it has been found that when Asp is replaced by GGC, it lowers the rate of replication and this mutation occurs due to mutated tRNA. In the case of âˆ†mutS there is no change as the codon codes for the same amino acid serine.