A plasmid vector is an autonomously replicating material which is extrachromosomal. E. coli plasmid vectors are common. The rolling circle model of replication is a well-known example. Plasmids get transferred to other cells. Bacterial conjugation involves the exchange or a direct transfer of F-plasmid known as fertility plasmid. It gets transferred from a donor to a recipient bacterium. Fertility plasmid allows DNA to pass through the cell. Hence, plasmids involve important functional parts of the bacterial genetic machinery. Replication in other cells is possible for the plasmids. Recombinant DNA technology involves cloning a specific gene.
Plasmids are widely used vectors in recombinant DNA technology. The technique involves inserting a fragment into a plasmid vector and transferring it to the host. Just like a vehicle helps in the transportation of goods from one location to another, plasmid vectors transport the DNA from one cell to another cell. Once the host transforms with the plasmid vector, it replicates the plasmid and gives the desired product. Some of the plasmids exchange small gene segments with the chromosomal material. They produce recombinants by integrating into the host genome.
Plasmids are widely used vectors in recombinant DNA technology. The technique involves inserting a fragment into a plasmid vector and transferring it to the host. Just like a vehicle helps in the transportation of goods from one location to another, plasmid vectors transport the DNA from one cell to another cell. Once the host transforms with the plasmid vector, it replicates the plasmid and gives the desired product. Some of the plasmids exchange small gene segments with the chromosomal material. They produce recombinants by integrating into the host genome.
Image: Plasmid Vector
Restriction sites in plasmids can be cleaved using specific restriction enzymes. However, they have limited number of restriction sites. The plasmids not only code for antibiotic resistance but also encode detoxification, virulence, and other interactions. The plasmid mobility and conjugation involves a separate set of genes. Nonmobilizable plasmids spread from transformation or transduction. A protein involved in the initiation and termination of direct gene transfer is known as relaxase. Direct gene transfer mediates through F-plasmid and resembles rolling circle replication components. Thus, the mobility of plasmids is a controlled mechanism. Since the plasmid DNA is double-stranded and circular, certain nicking proteins initiate the gene transfer in a process known as conjugation.
Following are the features of a plasmid vector:
An E.coli plasmid vector consists of an origin of replication. It is a point of initiation of replication. Hence an origin is required to start the replication process. One strand of the double-stranded DNA gets nicked to initiate the replication. A plasmid also consists of a selectable marker gene and a restriction site or a multiple cloning sites. The selectable marker exhibits traits that help us in selecting it. For example, resistance genes act as selectable markers. They are dominant. Suppose we culture two types of bacteria in the same medium. One type of strain transformed with a selectable marker containing a plasmid vector and other strain lacks a plasmid vector or the selectable marker. The cells lacking the plasmid or an antibiotic resistance gene accept the plasmid from the cells having the same. Hence, the direct gene transfer process involves the growth of only those bacteria that show the presence of a plasmid. Apart from an origin of replication and a selectable marker, other genes such as restriction sites are also present in a plasmid. These sites are enzyme specific.
Extraction of plasmids from a bacterial cell:
Bacterial cells are grown in the culture medium and then harvested and lysed to obtain pure samples of the plasmid DNA. Minipreparation is a rapid way to isolate the plasmids. Obtaining pure samples of the gene is possible with the plasmid vectors.
Vectors based on E. coli plasmids:
1. pBR322:
It is the first E. coli plasmid vector used in molecular biology. The letter “p” indicates plasmid and the letters “B” and “R” indicate Boliver and Rodriguez respectively. pBR322 has two antibiotic resistance genes. Bla gene indicates ampicillin resistance and tetA indicates tetracycline resistance. pBR322 exhibits unique restriction sites for HindIII and Cla I. Three naturally occurring E. coli plasmids including R1, R6.5, and pMB1 were involved in pBR322 construction. pBR322 vectors were involved in the derivation of pUC series of vectors.
2. pUC8 vector:
The pUC 8 vector is a small plasmid with a size of 2.7 kilobases. It has replication origin, a lac z’ gene, and ampicillin resistance gene. Lac z’ gene consists of a unique cluster of restriction sites for EcoRI, Sma I, Xma I, Bam HI, Sal I and many other sites. The presence of ampicillin resistance gene enables beta-lactamase synthesis. Presence of beta-lactamase ensures protection from the growth inhibitory effect of the antibiotic. Plating of the bacterial cells on an agar medium consisting of ampicillin helps distinguish plasmid. Normal E. coli cells lacking pUC 8 are sensitive to ampicillin and cannot grow. Those cells showing the presence of ampicillin resistance gene grew on the agar medium. Lac z’ gene encodes a beta-galactosidase enzyme. The presence of this enzyme enables the conversion of glucose to galactose.
Cell suspension in calcium chloride enables a better uptake of the plasmid vector. On insertion of a DNA fragment into the restriction site, insertional inactivation of the gene occurs leading to a loss of beta-galactosidase activity. Thus, a key to distinguish a recombinant plasmid from a non-recombinant plasmid is the presence of new DNA and loss of beta-galactosidase activity. A histochemical test involving X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) detects the presence or absence of the enzyme. The enzyme converts into a blue colored product. Addition of X-gal to an agar medium containing ampicillin made the non-recombinant colonies blue colored. It means they synthesized beta-galactosidase. Recombinant colonies appeared white. Hence, the system of Lac selection enables the distinction of recombinants from non-recombinants.
Cell suspension in calcium chloride enables a better uptake of the plasmid vector. On insertion of a DNA fragment into the restriction site, insertional inactivation of the gene occurs leading to a loss of beta-galactosidase activity. Thus, a key to distinguish a recombinant plasmid from a non-recombinant plasmid is the presence of new DNA and loss of beta-galactosidase activity. A histochemical test involving X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) detects the presence or absence of the enzyme. The enzyme converts into a blue colored product. Addition of X-gal to an agar medium containing ampicillin made the non-recombinant colonies blue colored. It means they synthesized beta-galactosidase. Recombinant colonies appeared white. Hence, the system of Lac selection enables the distinction of recombinants from non-recombinants.
3. pUC 19 vector:
It is a 2686 base pair vector derived from E. coli. It is a cloning vector with a high copy number. The vector has an ampicillin resistance gene as a selectable marker. Unique restriction sites or multiple cloning sites on the lac z’ gene have restriction sites for various enzymes. They are also known as polylinker sites. The Lac z’ gene encodes for beta-galactosidase. The N-terminal amino acid of the enzyme known as the alpha fragment is from the 5’ end. The plasmid constructed with a lac z’ gene lacking the short N-terminal expresses the truncated beta-galactosidase known as omega fragment which is inactive. The expression of both alpha and omega fragment in a cell dominates the expression of the alpha fragment. Hence it follows alpha complementation, meaning the expression of the wild-type dominates over the mutant. The expression of both the wild-type and mutant genes in a cell results in a wild-type phenotype of the progeny. Hence the interpretation follows the presence of complementation. Screening the pUC 19 vector involves lac selection. It is known as blue-white screening. Addition of X-gal to the agar medium enables identification of recombinants and the non-recombinants. Cells producing beta-galactosidase appear blue and the cells lacking beta-galactosidase appear white.
Insertion of DNA into pUC 19 plasmid vector:
A restriction enzyme is used to cut the polylinker site. Next step involves treating the desired DNA with restriction enzymes to obtain fragments of different sizes. Next step involves mixing the DNA fragments, the cloning vectors, and the ligating enzymes. A suitable fragment gets inserted into the vector and ligase seals the gaps. The so-called recombinant plasmid gets transformed into E. coli enhanced by chemical treatment or electroporation. Plating the cells on a suitable medium enables blue-white colony screening. Restriction mapping enables confirmation of the plasmid.
Multicopy plasmid vectors:
Multiple copies of these plasmids vectors in the cells express high levels of cloned genes. The ColE1 plasmid is a small circular DNA present up to 40 copies per cell. They are highly beneficial for transformation experiments. The colE1 plasmids consist of undesirable genes such as colicin genes. These genes produce a product that kills the bacteria. Thus, a transformation experiment involves the removal of colicin genes and the addition of ampicillin resistance genes.
References:
[1] Gene cloning and DNA analysis, T.A. Brown
[2] Biotechnology, R.C. Dubey
[3] Genetic Engineering, Anil Kumar, Neha Garg
[4] Molecular Biology, David P. Clark, Nanette J. Pazdernik
References:
[1] Gene cloning and DNA analysis, T.A. Brown
[2] Biotechnology, R.C. Dubey
[3] Genetic Engineering, Anil Kumar, Neha Garg
[4] Molecular Biology, David P. Clark, Nanette J. Pazdernik
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