The Extrachromosomal DNA - Essay Example

?Plasmid Analysis Plasmid DNA is the extrachromosomal DNA present in the bacteria. This plasmid DNA is able to carry the insert genes with it, and these genes are able to express themselves in the bacteria. When a gene is inserted into the Plasmid DNA, it is called Recombinant DNA. Restriction mapping is the process of cutting the Plasmid DNA using the restriction enzymes, and they also give us the idea whether the gene of interest is present in the correct position or not. White plasmid DNA was found to contain three restriction sites, of which two are for Hind III, and one site is for Sac I. The blue plasmid DNA did not contain any of the restriction sites for Hind III or Sac I. Introduction Plasmids are the extra chromosomal DNA molecules which are mostly double –stranded, circular and covalently closed molecules, varying in size from 1 kb to 200 kb. (Sambrook and Russell 2001). They are found in many bacterial species. They replicate independently and use a variety of mechanisms to maintain their copy number. They contain the gene codes for the enzymes that are important for the bacterial hosts. The plasmids act as vectors in the molecular biology experiments. The vectors are the carrier DNA molecules into which the foreign gene of interest is inserted and expressed in the host. This DNA is now called recombinant DNA (Roberts and Murray 1976). This recombinant DNA is able to express the Foreign DNA in the bacteria. These vectors replicate inside the host cell along with the inserted DNA. These vectors are of two types: expression vectors (expression of the cloned gene to give the desired protein) and cloning vectors (produce millions of copies of cloned DNA) (Sambrook and Russell 2001). Restriction endonucleases are the enzymes that cut the DNA at the specific sequences. There are about 200 different restriction enzymes (Siwach and Singh 2007). The pieces of DNA that remain after the digestion with the restriction enzymes are called restriction fragments. Each restriction enzyme has a unique code, and it cuts the DNA into fragments with either sticky or blunt ends (Brown 1995). A restriction map gives us the location where the restriction enzyme cuts the DNA. This restriction digestion is used for two purposes: restriction mapping and specific DNA cleavage for the production of new constructs. The restriction mapping is used to identify the plasmids. The number of DNA fragments and the size of the DNA fragments depend upon the action of the restriction enzyme (Kruezer and Massey 2008). These DNA fragments thus obtained are separated using the Agarose gel electrophoresis. Restriction mapping consists of three important steps. They are restriction enzyme digestion, agarose gel preparation and sample loading (Kruezer and Massey 2008). Results and Discussion: The nutrient agar plate was inoculated with E.coli, and the antibiotic discs were placed in the four quadrants. Figure1: Antibiotic profile against tetracycline in E. coli DH5alphaE:: pMTL84445 After inoculation at 37 degree Celsius for overnight, it was observed that the antibiotic disc of tetracycline had a clear zone. This indicates that the E.coli culture is resistant to kanamycin, chloramphenicol and ampicillin. There is very little sensitive to tetracycline. Figure 2: Antibiotic resistance profiling: Table 1a : Antibiotic resistance profiling of kanamycin control Kanamycin control E. coli DS941::pRRK Antibiotic disc Zone diameter in mm Chloramphenicol 30 Kanamycin 0 Tetracyline 10 Ampicillin 0 E.coli DS941::pRRK bacteria was found to be very sensitive to Chlormaphenicol and comparatively sensitive for Tetracycline antibiotics. The bacteria showed resistance to kanamycin and Ampicillin. Table 1b : Antibiotic resistance profiling of chloramphenicol control Chloramphenicol control: E. coli DS941::pAV35 Antibiotic disc Zone diameter in mm Chloramphenicol 0 Kanamycin 27 Tetracyline 32 Ampicillin 0 E. coli DS941::pAV35 bacteria were found to be very sensitive to Kanamycin and Tetracycline and resistant to Chloramphenicol and Ampicillin. Table 1c : Antibiotic resistance profiling of Ampicillin control Ampicillin control: E. coli DS941::pUC19 Antibiotic disc Zone diameter in mm Chloramphenicol 33 Kanamycin 16 Tetracyline 32 Ampicillin 0 Table 1d : Antibiotic resistance profiling of Tetracycline control E. coli DS941::pUC19 with ampicillin control showed resistance to ampicillin because of the presence of the resistance gene and showed sensitivity to the other three antibiotics such as Kanamycin, Chlormaphenicol and Tetracycline. Tetracycline control: E. coli DS941::pMTL84445 Antibiotic disc Zone diameter in mm Chloramphenicol not available Kanamycin not available Tetracyline 0 Ampicillin not available In the tetracycline controlled E.coli DS941::pMTL84445, there was resistance to all the antibiotics. Table 1e : negative control of Antibiotic resistance profiling Negative control: E. coli DS941 Antibiotic disc Zone diameter in mm Chloramphenicol 33 Kanamycin 27 Tetracyline 35 Ampicillin 30 The negative control of E.coli DS941 proved to be sensitive to all the antibiotics. Table 1f: Antibiotic resistance profiling for E.coli DS941: pB1 E. coli DS941::pB1 Antibiotic disc Zone diameter in mm Chloramphenicol 32 Kanamycin 21 Tetracyline 33 Ampicillin 0 For the E.coli DS941::pB1 bacteria, the resistance was observed only to ampicillin, and sensitivity to chlormaphenicol, Kanamycin and Tetracycline was found. Table 1g: Antibiotic resistance profiling for E.coli DS941: pW2 E. coli DS941::pW2 Antibiotic disc Zone diameter in mm Chloramphenicol 0 Kanamycin 17 Tetracyline 34 Ampicillin 0 Similarly, the E.coliDS941: pW2 showed resistance only to ampicillin. Table 2: Distance travelled by the plasmid vs DNA ladder Size ( kb) Marker Undigested Plasmid DNA white cut with Hind III white cut with Sac I white cut with Hind III and Sac I blue cut with hind III blue cut with Sac I Blue cut with Hind III and Sac I Undigested plasmid DNA 23.1 2 2 3.9 4.1 4.2 4.2 4.2 2 9 2.4 2.4 7.4 3.7 7.4 2.4 6.6 2.8 2.8 8.3 2.8 2.3 4.5 4.5 4.5 2.0 4.7 4.7 4.7 1.3 5.7 5.7 5.7 1.0 6.3 6.3 6.3 0.8 6.8 6.8 6.8 0.6 7.4 7.4 7.4 0.5 8.0 8.0 8.0 0.2 9.0 9.0 9.0 Figure 2: Restriction mapping of the plasmids: From the given gel, it was identified that the white cut with Hind III had two fragments of size 3.7kb and 0.6kb, white cut with Sac I produced one single plasmid DNA of size 4.3kb, white cut with Hind III and Sac I produced three fragments of size 3.3 kb, 0.6 kb and 0.4 kb, blue cut with hind III, and Sac I alone and in combination produced only one single band of size 2.8k, 2.8kb and 2.6kb respectively. The white cut with the Hind III had two fragments, and white cut with Sac I had one fragment. When the white was cut with the two restriction enzymes, three fragments were produced indicating that there is one restriction site for Sac I enzyme and two restriction sites for Hind III enzyme. The fragment sizes of the three fragments are 3.3, 0.6 and 0.4 kb respectively. This indicates that one restriction site for Hind III and Sac I is present nearer to each other, and the other restriction site for Hind III is a bit far away. The blue cut with the Hind III produced only one band, and the blue cut with Sac I also produced one band of the same size. When the blue was cut with the Hind III and Sac I, only a single band of the size similar to that of the other two restriction digests was observed. Since all the bands were of the same size, it was concluded that there is no restriction site for Hind III and Sac I in the blue plasmid. Figure 3: Standard curve for Hind III DNA fragments: The DNA fragments were cut by Hind III restriction enzyme at two parts of the plasmid DNA, so we get only two points in the logarithmic graph. For the blue cut with Hind III, no fragments were observed, so only one plot is available for this experiment. Table 2: Band size of the digests: S.No Restriction Digest Sample Band 1 size (kb) Band 2 size (kb) Band 3 size (kb) 1 WH 3.7 0.6 2 WS 4.3 3 WSH 3.3 0.6 0.4 4 BH 2.8 5 BS 2.8 6 BHS 2.6 Figure 4: Restriction Map of the Plasmid DNA White colony: When the white was cut with the Hind III and Sac I, three bands were formed. Of these three bands, one was of size 3.3kb and the other two were of size 0.6 kb and 0.4 kb respectively. Hence the restriction mapping will be as above. (Passarage 2007). References Brown, TA, 1995, Gene Cloning : An Introduction, Chapman and Hall, New York. Kruezer , H, and Massey, A, 2008, Molecular Biology and Biotechnology: a Guide for Teachers, ASM Press, London. Sambrook, J, and Russell, DW, 2001, Molecular Cloning: A Laboratory Manual, CSHL Press, London. Passarage, E., 2007, Color Atlas of Genetics, Thieme, London. Siwach, P, and Singh, N, 2007, Molecular Biology: Principles and Practice, Firewall Media, New York. Robert, RJ and Murray, K, 1976, “Restriction Endonucleases.” Critical reviews in Biochemistry and Molecular biology, Vol.4, no.2, pp.123- 164. Read More