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The cornerstone of most molecular biology technologies is the gene. To facilitate the study of genes, they can be isolated and amplified. One method of isolation and amplification of a gene of interest is to clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells. When these two DNAs of different origin are combined, the result is a recombinant DNA molecule. The recombinant DNA molecule is placed in a host cell, either prokaryotic or eukaryotic. The host cell then replicates (producing a clone), and the vector with its foreign piece of DNA also replicates. The foreign DNA thus becomes amplified in number, and following its amplification can be purified for further analysis.
Vectors are used to compile a library of DNA fragments that have been isolated from the genomes of a variety of organisms. This collection of fragments can then be used to isolate specific genes and other DNA sequences of interest. DNA fragments are generated by cutting the DNA with a specific restriction endonuclease. These fragments are ligated into vector molecules, and the collection of recombinant molecules is transferred into host cells, one molecule in each cell. The total number of all DNA molecules makes up the library. This library is searched, that is screened, with a molecular probe that specifically identifies the target DNA. Once prepared the library can be perpetuated indefinitely in the host cells and is readily retrieved whenever a new probe is available to seek out a particular fragment.
Two major categories of enzymes are important tools in the isolation of DNA and the preparation of recombinant DNA: restriction endonucleases and DNA ligases. Restriction endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere, depending on the type of enzyme. DNA ligase joins two pieces of DNA by forming phosphodiester bonds.
There are three major classes of restriction endonucleases. Their grouping is based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure. Type I and III restriction endonucleases are not useful for gene cloning because they cleave DNA at sites other than the recognition sites and thus cause random cleavage patterns. In contrast, type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites. In addition, the type II endonuclease and methylase activities are usually separate, single subunit enzymes. Although the two enzymes recognize the same target sequence, they can be purified separately from each other.
Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are that they: (i) can independently replicate themselves and the foreign DNA segments they carry; (ii) contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector; (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host cells that do not contain a vector; and (iv) are relatively easy to recover from the host cell. There are many possible choices of vector depending on the purpose of cloning. The greatest variety of cloning vectors has been developed for use in the bacterial host E. coli. Thus, the first practical skill generally required by a molecular biologist is the ability to grow pure cultures of bacteria.
The classic cloning vectors are plasmids, phages, and cosmids, which are limited to the size insert they can accommodate, taking up to 10, 20, and 45 kb, respectively. A cosmid is a plasmid carrying a phage ë cos site, allowing it to be packaged into a phage head. Cosmids infect a host bacterium as do phages, but replicate like plasmids and the host cells are not lysed. Mammalian genes are often greater than 100 kb in size, so originally there were limitations in cloning complete gene sequences. Vectors engineered more recently have circumvented this problem by mimicking the properties of host cell chromosomes. This new generation of artificial chromosome vectors includes bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and mammalian artificial chromosomes (MACs).
Plasmids are naturally occurring extrachromosomal double-stranded circular DNA molecules that carry an origin of replication and replicate autonomously within bacterial cells.
What needs to be included in the medium for plating cells so that nontransformed bacterial cells are not able to grow at all? The answer depends on the particular vector, but in the case of pUC18, the vector carries a selectable marker gene for resistance to the antibiotic ampicillin. Ampicillin, a derivative of penicillin, blocks synthesis of the peptidoglycan layer that lies between the inner and outer cell membranes of E. coli. Ampicillin does not affect existing cells with intact cell envelopes but kills dividing cells as they synthesize new peptidoglycan. The ampicillin resistance genes carried by the recombinant plasmids produce an enzyme, â-lactamase, that cleaves a specific bond in the four-membered ring (a-lactam ring) in the
ampicillin molecule that is essential to its antibiotic action. If the plasmid vector is introduced into a plasmidfree antibiotic-sensitive bacterial cell, the cell becomes resistant to ampicillin. Nontransformed cells contain no pUC18 DNA, therefore they will not be antibiotic-resistant, and their growth will be inhibited on agar containing ampicillin. Transformed bacterial cells may contain either nonrecombinant pUC18 DNA (selfligated vector only) or recombinant pUC18 DNA (vector containing foreign DNA insert). Both types of transformed bacterial cells will be ampicillin-resistant.
To distinguish non-recombinant from recombinant transformants, blue-white screening or "lac selection" (also called áa-complementation) was used with this particular vector. Bacterial colonies were grown on selective medium containing ampicillin and a colorless chromogenic compound called X-gal, for short (5-bromo-4-chloro-3-indolyl-â-d-galactoside). pUC19 carries a portion of the lacZ gene (called lacZ â€²) that encodes the first (last) 146 amino acids for the enzyme â-galactosidase. The multiple cloning site resides in the coding region. If the lacZ â€² region was not interrupted by inserted DNA, the amino terminal portion of a-galactosidase was synthesized. Importantly, an E. coli deletion mutant strain was used
(e.g. DH5á) that harbors a mutant sequence of lacZ that encodes only the carboxyl end of â-galactosidase (lacZâ€² ÄM15). Both the plasmid and host lacZ fragments encode nonfunctional proteins. However, by a-complementation the two partial proteins could associate and form a functional enzyme. When present, the enzyme a-galactosidase catalyzed hydrolysis of X-gal, converting the colourless substrate into a blue-colored product.
Two commonly used plasmid vectors are shown above These were artificially
constructed for the purpose of isolating and moving pieces of foreign DNA into microbial
systems. pBR322 contains the genes for ampicillin resistance and tetracycline resistance.
This means that a microorganism containing this plasmid is capable of growth on media
containing both of these antibiotics, while microorganisms that do not contain this
plasmid could not grow on the antibiotic containing media. This antibiotic resistance can
be used as a means of selecting and enriching for only those organisms that contain the
Escherichia coli XL-1 Blue/pUC18 host/vector system
Strains of Escherichia coli were grown in Luria-
Bertani media (LB) containing the appropriate
antibiotics at 37oC. A. tumefaciens strains were grown
at 28oC in mannitol glutamate luria salts medium (MG/
L). Gentamicin was filter-sterilized and added to E. coli
cultures at 25 ìg/mL and 50 ìg/mL of cultures. Sucrose
was added to media at a concentration of 5% to select
against plasmid carrying the Bacillus subtilis levansucrase
gene. X-gal was obtained from Research Product
Internation Corp. (IL 60056) and used at a
concentration of 50 mg/mL.
Abstract: Escherichia coli as a plasmid recipient cell
Escherichia coli strains, including JM109 (el4-,
recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1,
Î”(lac-proAB), [F', traD36 proAB, lacIq ZÎ”M15]),
were inoculated into Luria-Bertani (LB) agar plate
medium or LB broth (Sambrook et al. 1989) and
incubated or shaken for 15-18 h at 37Â°C.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning:
a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, New
York: Cold Spring Harbor.
Fig.2Â Â Â Â Â Â SDS-PAGE of total protein in transformants containing argS and argSDn
Molecular weight markers from Sigma, with molecular weight of 97.4, 66.0, 45.0, 31.0, 21.5 and 14.5 kD (lane 1); purified ArgRS (lane 2); proteins in the crude extract of TG1 transformants containing argS (lane 3), argSD8 (lane 4), argSD7 (lane 5), argSD6 (lane 6), argSD5 (lane 7), argSD4 (lane 8), argSD3(lane 9), argSD2 (lane 10), argSD1 (lane 11); proteins in the crude extract of TG1 (lane 12). In each lane 15 ml of sample was loaded.
XL1-Blue supercompetent cells transformed
with this control plasmid appear white on LB-ampicillin agar plates (see
Preparation of Media and Reagents), containing IPTG and X-gal, because
Î²-galactosidase activity has been obliterated.
E. coli XL1-Blue was grown at 37Â°C with agitation in Luria-Bertani broth (LB) supplemented with 10 Âµg/mL tetracycline. When E. coli XL1-Blue was transformed by pUC18 or recombinant plasmids, a 50Âµg/mL quantity of ampicillin was added to the medium. E. coli XL1-Blue bearing the p517spec and p517specbla plasmids was grown in LB medium supplemented with 100 Âµg/mL spectinomycin. S. mutans NDBX-10 was grown in TYE supplemented with 0.2% (w/v) glucose and 600 Âµg/mL spectinomycin.
When one wants to genetically modify an organism, a so-called vector is usually used for the process. The usage of plasmids, a type of vector, is most frequent, which are small double stranded DNA molecules. The transfer operation of these plasmids into a bacterium's chromosome, is fairly easily done. Plasmids account for about 5% of the bacterial genome, and they are able to replicate outside the bacterium's chromosome. The chromosome features, amongst other things, the genes, that make it resistant to a number of chemicals.
In eucaryote cells, plasmids come to pass very seldom. So-called restriction enzymes, make the insertion, of a desired gene in to a plasmid, possible. They identify specific nucleotide DNA molecules, usually of 4 to 6 bp segment and cleave the strand within those sequences. The same restriction enzyme specifically applies to the plasmid, as to the, becoming, inserted DNA strand. The both ends of open circular plasmid are complimentary to both ends of the DNA segment, respectively. The segment is inserted into the circle with the help of intergrating enzymes.
In this practical experiment, R21-segment was integrated, via HindIII-restriction site, into the pUC18 plasmid (2700 bp), subsequently called pUC18-R21. Then, pUC18-R21, was cleaved, separately, by four restriction enzymes, and the segments formed, were electrophoresised in agarose gel, and finally the results photographed (FIGURE 02 â- ). The enzymes used, cleave the pUC18 plasmid, just once or never (XhoI).