Plasmids are circular, self-replicating, double -stranded extra chromosomal DNA molecules. E. coli plasmids can be tailored for its extensive use as cloning vectors in Recombinant DNA technology because of its genetic simplicity. (Sandy. B Primrose, Richard M Twyman, Robert W Old 2001) These plasmids usually have a high copy number, low molecular weight, ability to render selectable phenotypic traits and a single site can incorporate a number of restriction endonucleases. In addition to these essential constituents required for cloning, plasmids also contain a replication origin, an antibiotic resistance gene and a region in which the exogenous DNA can be inserted. (Lodish, 2008)
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AIM AND OBJECTIVES:
To start with the subcloning of PstI fragment from the pMB into pUC 19, the plasmids pMB and pUC 19 are digested with PstI restriction enzyme. The restriction fragment is then isolated from the pMB and the vector pUC 19 is treated with shrimp alkaline phosphatase to remove the 5’ends of the plasmid. The DNA is then purified using selective column binding followed by the ligation of the insert fragment and vector to produce the recombinant pUC19. In the meantime, competent E. coli XL1 blue (tetracycliner) competent cells are prepared and are tested for efficiency. These cells are then transformed with the DNA molecules and are eventually screened (blue/white X-gal screening) for recombinant bacterial colonies on agar plates (Ampicillin and IPTG induced). Finally, the recombinant bacterial colonies are picked to conduct a small scale plasmid preparation which successfully terminates the desired subcloning. The insert fragments can now be released by digesting the recombinant plasmid with PstI and analysed by electrophoresis.
- Molecular basis of a-complementation and blue/white selection:
The pUC plasmids are the most widely used cloning vectors with an easily selectable ampicillin resistance and the ease of insertional inactivation. It encodes for the N-terminal region of the LacZ gene (a-peptide) that complements the C-terminal of Lac Z ?-peptide turning the initially inactive one to active. This turns the colonies blue on X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside). Here the host chromosome codes for the remaining ?-peptides that produces the enzyme ß-galactosidase after tetramerization. This phenomenon is called a-complementation. (Nelson et al. 2008) When a foreign DNA is inserted at the Lac Za gene, the production of the ß-galactosidase enzyme gets inhibited (no production of a-peptide)and hence the production of white colonies. This is mainly due to the loss of the recombinant cells’ ability to hydrolyse the markers. The Lac region is usually inducible and is turned on by the use of IPTG (isopropyl-ß-D-galactosidase). (Griffiths 2007)
- Molecular basis of Ampicillin resistance:
Ampicillin is atype of ß-lactam antibiotic. A given bacterium is usually made antibiotic resistance by transduction, transformation, conjugation or fusion with the methods having its own limitaions. The objective is to genetically transfer a ß-lactamase encoding gene called the bla gene via plasmid transfer, if not present inherently in the bacterial chromosome, which on hydrolysis confers the resistance to the bacterium. (Atlas 1988) This is also mediated by transposition wherein the transposon (a DNA unit), can move within the same structure in a bacterial cell carrying the gene specific for an antibiotic resistance from one self-replicating DNA to another. In the case of ampicillin, a Tn3 unit confers resistance to it due to the presence of the bla gene. The structure usually moves as a whole, inducing changes in the genetic composition of the bacterium, offering resistance to ampicillin. This spread of ampicillin resistance is generally monitored at any of these three stages transposition step, an extrachromosomal unit (if the gene is present in the chromosome, celll-cell transfer) and at the point of infection. The ampicillin also competes for the Transpeptidase enzyme which aids bacterial cell wall synthesis and eventually leads to lysis. This inhibition is done by the target modification of the outer bacterial cell wall and the penetrability through the cell wall is varied to accommodate other essential metabolites too. (Gale 1981)
The strains of pMA and pMB with three others (DH5a, XL1 blue, pUC 19) are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. pMA is found to be both ampicillin and tetracycline resistant whereas pMB is found to be tetracycline and kanamycin resistant. The PstI fragment subcloned from pMB contains Kanamycin as an antibiotic resistance, this cannot be tetracycline because when this antibiotic meets the cell wall of a sensitive bacteria (pUC19-amp resistant) it has an initial free inflow and outflow which is later followed by a block of the outflow leading to accumulation of the tetracycline within the cell. To optimize on its flow into the cell, there must be an alteration in the peripheral membrane proteins which require suitable induction. (Gale 1981)
Competent E. coli XL1-blue cells were prepared and stored at -800c after the addition of glycerol. The transformation efficiency of the competent cells were calculated.
- 50µl of 1ng pUC19 DNA: 8. 2*106
- 50µl of 5ng pUC19 DNA: 1. 94*106
- 200µl of 1ng pUC19 DNA: 7. 25*106
- 200µl of 5ng pUC19 DNA: 1. 82*106
Gel purification of both the insert fragment and the PstI-cut pUC19 vector DNA were carried out and ligated. The ligated samples were transformed with the competent cells to grow recombinant cells on LB agar plates containing ampicillin, IPTG and X-gal. (Huff et al. 1990)
Small scale plasmid preparation of the potential subclones was set up and the restriction analysis of plasmid DNA was carried out by releasing the insert fragment from the vector by digesting the recombinant plasmid with PstI.
Hence the PstI fragment from the pMB got successfully subcloned into the pUC19 which is shown by the similar sizes of the insert into the vector DNA molecule during the small scale preparation of the plasmid. Here a Positive selection could have been employed for recovering only bacterial clones with recombinant pUC19 containing the PstI fragment from pMB. This allows only the recombinant clones and masks the growth of the wild-type organism. Otherwise, the use of Selected and Unselected markers can provide similar results with the selected marker to spot the recipient vector DNA that has undergone recombination and also look for other markers they can mutate with and the other markers can be now termed unselected markers. (Snyder & Champness 2007). The chain of experiments instilled in us the required hands-on experience required for the preparation of recombinant clones in a systematic order.
- Atlas, R. M., 1988. Microbiology: fundamentals and applications. 2nd ed. New York; London: Macmillan; Collier Macmillan.
- Gale, E. F., 1981. The molecular basis of antibiotic action. 2nd ed. London: Wiley.
- Griffiths, A. J. F., 2007. Introduction to genetic analysis. 9th ed. New York: W. H. Freeman.
- Hartwell, L., 2007. Genetics: from genes to genomes. 3rd ed. Boston, Mass. ; London: McGraw-Hill Higher Education.
- Huff, J. P., Grant, B. J., Penning, C. A. and Sullivan, K. F., 1990. Optimization of routine transformation of Escherichia coli with plasmid DNA. BioTechniques, 9, 570-2, 574, 576-7.
- Lodish, H., 2008. Molecular cell biology. 6th ed. New York: W. H. Freeman.
- Nelson, D. L., Cox, M. M. and Lehninger, A. L., 2008. Lehninger principles of biochemistry. 5th ed. New York, N. Y. ; Basingstoke: W. H. Freeman.
- Sandy. B Primrose, Richard M Twyman, Robert W Old, 2001. Principles of Gene Manipulation sixth edition ed. United Kingdom: Blackwell Science Ltd.
- Snyder, L. & Champness, W., 2007. Molecular genetics of bacteria. 3rd ed. Washington, D. C. : Asm.
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