The Determination of Antibiotic Resistance and Restriction Sites in Plasmids in Transformed Bacterial Cells - Lab Report Example

The Determination of Antibiotic Resistance and Restriction Sites in Plasmids in Transformed Bacterial Cells Recombinant DNA technology isused in the cloning of DNA that code for genes of interest in various organisms. This process has seen the production of numerous recombinant products such as antibiotics, enzymes and hormones. Bacterial plasmids are one of the commonly used vectors in this process. Antibacterial resistance is one of the selective markers that are used to select for transformation. This practical aimed at determining the antibiotic resistances conferred on four E. coli strains (PAV35, PRRK, MTL84422 and DH5 ἀ) that harboured two plasmids with foreign DNA inserts (white and blue plasmids). It also intended to generate restriction maps for three restriction enzymes in the two plasmids. It was shown that only PAV35 contained the recombinant white plasmid. It was also revealed that the blue plasmid contained one restriction site for SacI and HindIII, whereas the blue plasmid contained two restriction sites for HindIII and one for SacI. Introduction A series of processes that are employed in joining fragments of DNA is called recombinant DNA technology (Khan 2011). Such processes enable the combination of different pieces of DNA from different organisms to create a novel gene that is otherwise unavailable in nature. The similarities in the basic chemical structure of DNA in all living organisms make it possible for such procedures to be performed and have seen tremendous success in gene cloning and manipulation. The foreign DNA is inserted into the gene of interest using artificial carriers known as vectors (Griffiths et al. 2000; Wong 2007). Some of the commonly employed vectors are bacterial plasmids, yeast artificial chromosomes, cosmids, and bacteriophages (Nair 2005). One of the most important traits of cloning vectors is the possession of suitable restriction enzyme sites to facilitate the insertion of the DNA of interest (Alberts et al. 2013). In addition, they ought to have selectable markers to enable the identification of host cells that have taken up the foreign DNA (Recombinant DNA technology n.d). One such marker is antibiotic resistance. For example, when a penicillin-resistant plasmid is used in cloning, only cells that have taken up the plasmid can grow in penicillin thereby enabling the identification and selection of the recombinant cells (Thomas 2013). This practical intended to determine the antibiotic resistances conferred on bacteria that harboured the two plasmids with foreign DNA inserts. It also aimed at determining the map of sites for three restriction enzymes in the two plasmids. It was hypothesised that there was a relationship between the antibiotic resistance of a plasmid and a bacterial cell that took up that plasmid (null hypothesis was that there was no relationship between the antibiotic resistance of a plasmid and bacteria that took up the plasmid). Methods As per the protocol. Results Bacterial plates In the white plasmid plate, bacteria grew on ampicillin and chloramphenicol but not on kanamycin and tetracycline. In the blue plasmid plate, bacteria grew only on ampicillin but not on kanamycin, tetracycline and chloramphenicol. For plates MT84422, only tetracycline allowed bacteria to grow but not chloramphenicol, ampicillin and kanamycin. For plate PRRK, only chloramphenicol inhibited bacterial growth. For plate PAV35, ampicillin and chloramphenicol allowed bacteria to grow while kanamycin and tetracycline did not. For plate DH5ἀ, bacteria did not grow on any of the discs. These results are shown in figures 1 and 2. Figure 1: Bacterial plates showing the growth of bacteria in the presence of various antibiotics. The first two plates at the top are DH5 ἀ and DH5: ἀ pRRK(KanR). The second pair of plates at the bottom are DH5 ἀ: MTL84422 (tetR) and DH5: ἀ pAV35 (ampR/CamR). Figure 2: The controls of the bacterial antibiotic resistance. The plate on the left shows the DH5 ἀ: p Blue while the plate on the right shows DH5 ἀ: p White Antibiotic Resistance Profiles Zones of Inhibition Table 1: White plasmid plate Antibiotics Zones of inhibition in cm Ampicillin 0 Chloramphenicol 0 Kanamycin 2 Tetracycline 3 Table 2: Blue plasmid plate Antibiotics Zones of Inhibition Ampicillin 0 Kanamycin 2.1 Tetracycline 2.7 Chloramphenicol 2.5 Table 3: Plate PRRK Antibiotics Zones of inhibition Ampicillin 0 Chloramphenicol 2.7 Kanamycin 0 Tetracycline 0 Table 4: Plate PAV35 Antibiotics Zones of inhibition Ampicillin 0 Chloramphenicol 0 Kanamycin 2.9 Tetracycline 2.6 Table 5: Plate MTL84422 Antibiotics Zones of inhibition Ampicillin 1.3 Chloramphenicol 1.6 Kanamycin 2.8 Tetracycline 0 Table 6: Plate DH5 ἀ Antibiotics Zones of inhibition Ampicillin 2.1 Chloramphenicol 2.7 Kanamycin 2.0 Tetracycline 2.5 Table 7: A summary of the antibiotic resistance profiles of the various plasmids Antibiotic Plasmid White Blue PRRK PAV35 MTL84422 DH5 ἀ Ampicillin + + + + - - Chloramphenicol + - - + - - Kanamycin - - + - - - Tetracycline - - + - + - Key: + = resistant; -= susceptible Gel Photo Figure 3: A photo showing the bands obtained in agarose gel electrophoresis of the plasmid DNA digested by various restriction enzymes. Lanes 1, 2 and 9 are DNA molecular ladders. Lane 3 shows white plasmid cut with HindIII, lane 4 shows white plasmid cut with SacI, and lane 5 shows white plasmid cut with HindIII and SacI. Lane 6 shows blue plasmid cut with HindIII, lane 7 shows blue plasmid cut with SacI and lane 8 shows blue plasmid cut with HindIII and SacI. Distance travelled by DNA Fragments Table 8: The distance travelled by various band sizes of DNA Distance migrated Band sizes 38 23.1 49 9.4 59 6.5 112 2.3 122 2 131 1.3 146 1 153 0.87 166 0.6 178 0.3 Standard Curve Figure 4: A standard curve for determining the size of DNA bands. Sizes of Fragments Table 9: The sizes of the bands obtained from the digest and their corresponding sizes (bp) Digest Distance travelled (mm) Bands created Size (bp) HindIII 80 and 142 2 4002.5, 1040.1 Sac1 74 1 4808.8 WHS 83,141 and 160 3 3671, 1057.5, 785.6 Blue plasmid HindIII 85 1 3471.3 SacI 86 1 3377.3 BHS 87 and 178 2 3286.8, 611.8 Where the plasmid had been cut up by two enzymes, the sum of the sizes of the individual bands did not add up correctly. Restriction Maps Figure 5: The restriction maps of the two plasmids Discussion It was observed that the white plasmid was resistant to ampicillin and chloramphenicol and was susceptible to kanamycin and tetracycline while the blue plasmid was resistant to ampicillin only and susceptible to chloramphenicol, kanamycin and tetracycline. Some of the plates had bacteria growing in the discs because they were resistant to the antibiotics in the discs. The antibiotic resistance genotype was present in the genes of these bacteria. The ability to grow in the presence of the antibiotics was the phenotypic expression of the presence of the antibiotic resistance genotypes in the bacteria. For example, the white, blue, PRRK and PAV35 plasmids contained the beta-lactamase genotype that enabled growth on ampicillin (Purdue University 1996; Tofteland et al. 2007). From the antibiotic resistance profiles of the various bacteria, it was shown that only PAV35 contained the white plasmid, which had the gene of interest. That was shown by the similarities in the antibiotic resistance profiles of the white plasmid and PAV35. These findings were consistent with numerous other studies that showed that white plasmids in the blue-white screening technique contained the foreign DNA insert (Chaffin & Rubens 1998; Wong & Truong 2010). DH5 ἀ, on the other hand, was susceptible to all the four antibiotics. It was, therefore, a good strain to be used in cloning using antibiotic resistance as a selectable marker since the introduction of an antibiotic resistance plasmid would confer antibiotic resistance to it. The other bacteria (PRRK, MTL84422 and DH5 ἀ) did not take up either of the white or blue plasmids. However, it was observed that where the plasmid had been cut up by two enzymes, the sum of the sizes of the individual bands did not add up correctly. That was probably because the digestion of the fragments did not take place completely (Brown, Knudson, & Ishimaru 2002). It was also possible that some small fragments, which were invisible in the gel were produced making it difficult to visualize them. Another possibility was that some of the small fragments ran of the gel during electrophoresis making it difficult for them to be detected (Chan et al. 2002). The molecular weight ladders in figure 3 gave different patterns just as was observed in a similar study by Gagnon (2005). The fragments in lane 1 differed from those observed in lane 2 and lane 9. In addition, some bands were more intense than others. This showed that there were some inconsistencies in the DNA migration patterns, which probably had an effect in the sizes of the digested fragments that were observed. The white plasmid was shown to have two restriction sites for HindIII and one restriction site for SacI, whereas the blue plasmid had one restriction site for HindIII and SacI. Summary and Conclusion It was realized that bacteria that took up a recombinant plasmid expressed the phenotypic traits of the plasmid. The results of this study, therefore, supported the alternative hypothesis that a relationship existed between the antibiotic resistance of a plasmid and a bacterial cell that took up that plasmid. It was concluded that plasmids were valuable media of conveying foreign DNA from one organism to another. 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C. & Truong, K 2010, “Fluorescent protein-based methods for on-plate screening of gene insertion,” PLoS ONE, vol.5 no.12, e14274. Read More