DNA Cloning is a method for replicating a DNA of interest. For its replication, it has to be inserted into a vector, usually a highly modified phage/plasmid that can replicate in a host cell.
Generally a plasmid vector contains three elements - a cloning site where a foreign DNA fragment can be inserted, a drug-resistant gene that destroys antibiotics to allow replication of the host cell eg. Ampicillin, and an origin of replication that allows the plasmid to replicate in the host cell. To insert foreign DNA, we should use a restriction enzyme to cleave the vector at the cloning site. The restriction enzyme cleaves the palindromic sequence to produce single stranded ends called sticky ends. These can hybridize with any piece of DNA that has also been cut by the same restriction enzyme.
Foreign DNA containing the sequence that is to be cloned is digested with restriction enzyme and then mixed with the cleaved vector. The sticky ends of foreign and plasmid DNA molecule hybridize and then are sealed at phosphodiester linkages by the enzyme DNA Ligase, creating a recombinant DNA. Each of these recombinant DNA contains the inserted DNA fragment, an antibiotic resistant gene and an origin of replication. A pool or library of circular recombinant DNA is thus created. Each plasmid carries a unique fragment of foreign DNA. Next, the recombinant DNA are added to the host cells, Escherichia Coli. Through a process called transformation, a few cells take up s recombinant plasmid, while other cells do not. The bacterial cells are passed onto a plate of agar containing the antibiotic, for instance ampicillin. With the antibiotic in the agar, only cells that are resistant to the drug can grow. They will produce a separate colony of cells as they cannot move about on the agar medium. The non-transformed cells lack the antibiotic resistance gene and die.
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The origin of replication, allows the plasmid to replicate by using enzymes of the host cell. Plasmid replication is independent of host cell division. But, plasmids are replicated to each daughter cell when the whole cell divides. As plasmids replicate and host cells multiply, the number of recombinant plasmids gets amplified to a great extent. The multiple daughter cells form a colony of clones because all host cells in a colony are derived from a single cell, they all contain copies of the same recombinant plasmid with its fragment of formed DNA. A variety of assay methods can now be used on bacterial colonies to determine which contains the particular target DNA sequence.
A fragment of Pst1 was isolated from pMB and transferred to pUC19. pUC19 and pMB were both treated with restriction enzyme Pst1. This resulted in sequences with sticky ends. Alkaline Phosphatase was added to the pUC19 to remove sticky ends. Competent E.Coli cells treated with CaCl2 were added to the pUC19 to make them permeable to the DNA molecules.
When two inactive fragments of the enzyme combine to form a functional protein, it is called a Complementation. This mechanism is used for determining whether the transformed bacterial cell has the plasmid with the target insert or not. And it can be best explained with regard to the Lac operon of the Escherichia Coli. Lac operon contains a gene called Lac gene which encodes for an enzyme namely b-Galactosidase. b-Galactosidase is a tetramer, made up of four identical monomers. ß-Galactosidase consists of an a-subunit at the N-terminal end and ?- subunit at the C-terminal end. The a and w subunits of the enzyme, even in their inactive form associate with each other and form a functionally active enzyme. The main function of the enzyme is to cleave lactose into glucose and galactose. And in this instance it is used to break down a compound called X-Gal (5-bromo-4-chloro-3-indolyl- b-D-Galactoside) into an insoluble blue product. The pUC family comprises only a part of the LacZ gene, called the LacZ', that encodes a part of the enzyme - the a peptide. The remaining portion of the LacZ gene is present in the w subunit in E.Coli. When the LacZ' associates with the LacZ in E.Coli, b-Galactosidase is tetramerised, resulting in an active b-Galactosidase enzyme that hydrolyses X-Gal. The hydrolysis causes the formation of 5-bromo-4-chloro indole, which turns blue. Whereas, if the LacZ gene present in the multiple cloning site of the pUC19 is inserted with a pMB fragment, then the LacZ' gene is replaced by the insert molecule and hence would not associate with the w subunit in E.Coli. This results in the absence of any b-Galactosidase function as it does not form a tetramer. Hence X-Gal would not be hydrolyzed and recombinant molecules would not be formed.
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Complementation can be easily understood using a screening method called Blue/White selection. When there is no insert fragment, the colonies will have an intact LacZ gene and will metabolize X-Gal into 5-bromo-4-chloro indole, that turns blue indicating that hydrolysis of X-Gal has taken place. Whereas the colonies that have an insert fragment would be unable to metabolize X-Gal and hence would remain white in colour (recombinant clones).
Ampicillin is an amino penicillin which belongs to a family of antibiotics called b-Lactam antibiotics. The antimicrobial activity of ampicillin extends upto gram negative and gram positive bacteria. Certain bacteria contain an ampicillin resistant gene, that is present wither in the chromosome or the plasmid of the bacteria. b-Lactamases are present in these bacteria. These b-Lactamases cleave the four membered cyclic ring of the b-Lactam, thus making the antibiotic inactive. Only bacteria that successfully take up the desired genes become ampicillin resistant, and therefore contain the other desired gene as well.
While testing for resistance in the bacterial colonies, it was inferred that pUC19 and pMA are resistant to Ampicillin and not resistant to Kanamycin. Whereas, pMB is non-resistant to Ampicillin and shows resistance to Kanamycin. Though ampicillin resistance would identify recombinant clones from non-recombinant ones, it would represent both of them. But if Kanamycin resistance is used, only recombinant clones will be present as the other cells would die. Hence, it would be more efficient, if Kanamycin resistance is used.