Screening of clones

Just like collecting books and arranging them in a library, the recombinant DNA library stores the cloned DNA fragments. For selecting a particular clone, we need to screen these libraries. Hence, before knowing the screening procedures, we must know the types of libraries. There are two main types of libraries such as cDNA library and genomic library. The cDNA library involves a collection of cloned cDNAs produced from a mRNA. The DNA sequence gets represented at least once in a genomic library.

1.   Screening a cDNA library:

For screening, we need to search for a cDNA clone encoding a protein. An antibody probe screens specific cDNA plasmid. It involves the use of an expression vector. Since we need to search for a cDNA clone encoding a protein, we use expression vectors. An expression vector is nothing but the vectors designed for the expression of the genes. When the cDNA gets cloned into an expression vector, the gene gets expressed and produces proteins. The cDNA gets inserted into an expression vector such that it gets fixed between the promoter and the termination signal. When cDNA gets inserted in the host, the gene gets expressed. The selection of the host involves E. coli cells. The expression vector containing cDNA gets inserted into the bacteria through the process of transformation. Why only expression vectors? An expression vector expresses the genes efficiently. Screening the cDNA clones requires a specific host such as E. coli. The growth of the cells helps us in identifying the recombinants and non-recombinants. It depends on the expression of the cDNA or the desired gene. Designing an expression vector depends on the type of organisms such as a prokaryote or a eukaryote. Hence, for every type of an organism, an expression vector design gets ready. Plus, we add a purification tag or a fusion partner along with the cDNA or a cloned gene. It helps in obtaining a purified product.

Plating the bacterial cells (transformed with the vector) on a selective medium allow the bacterial colonies to grow. Individual colonies get picked up and transferred to a microtiter dish. It is a 96 well dish. A microtiter well serves as a liquid holder. Several milliliters of liquid gets filled in the microtitre dish or well. Microtitre dish or a well has many applications. They serve in screening, filtration, separation, storage, culturing cells, detecting the anti-microbial activity, and many other uses. Microtiter dishes adjust with the temperatures. The evaporation of the solvent occurs more after heating.

Next step involves a pertiplate with a selective medium. A membrane filter placed on the Petri plate for getting the replicas of the clones. The selective medium is appropriate for the recombinant molecules. The pattern of the growth of colonies on the membrane filter is the same as the colonies growing on the microtiter wells. After observing or allowing growth of the colonies, the filter gets peeled from the dish. The cells get lysed using a lysis solution in situ. The protein product of cDNA gets bound to the filter. Next step involves incubation with an antibody labeled radioactively. The placement of the filter in the X-ray film helps in observing the expressed protein of interest. This process is also known as autoradiography. The development of X-ray film gives dark spots in the region where radioactive probe gets bound.

Image 1: Procedure for Screening of clones

Image 2: Antibody-Protein interaction

2.   Screening a genomic library:

The cloned gene coding for mRNA molecule gets identified using plasmid genomic libraries. The technique involves DNA probes. The construction of the genomic libraries depends on a vector such as a plasmid vector. The procedure for screening a genomic library is similar to that of the cDNA library. The host used in screening procedures involve E. coli bacterial cells. The first step involves the transformation of genomic libraries into E. coli cells. The cells plated on a selective medium help in obtaining the growth of the colonies. The technique involves replica plating. It helps in producing identical copies of a series of the bacterial colonies on Petri dishes. First, the Petri plate consisting of the bacterial colonies gets inverted. The surface of this dish gets pressed against a cylindrical block and with a velveteen cover. Hence, the bacterial colonies present on the Petri-plate get transferred to the velveteen. There is an extension to this procedure. The pressing of the velveteen consisting of the cells from the original colonies to a plate consisting of a selective medium with a membrane filter allows the cells to grow on the filter. After the cell growth, the membrane gets lifted and processed for cell lysing.

After the bacterial cell lysis, the DNA gets denatured into single-stranded structures. Then the DNA firmly binds to the filter. After placing the filter on a heat-stable bag, the cDNA probes get incubated with cDNA probes. The labeling of the probes with radioactive or a non-radioactive element involves separate steps. First, the DNA gets denatured by boiling. Next, a quick way of cooling it involves ice for producing the single-stranded molecules. The synthetically obtained primers get annealed to DNA. Next, pairing with the hexanucleotide primers takes place. They get elongated by Klenow fragment of DNA polymerase I using radioactively labeled precursors (dNTPs). An example includes P³² labels. A special DNA precursor molecule gets involved in the case of non-radioactive labeling. The precursor involves the use of the digoxigenin dUTP. It also involves usage of chemiluminescent substrates. The labeled DNA molecules get diffused over the filter. Complementary base pairing and hydrogen bonding form DNA-DNA hybrids. The formation of the hybrids occurs between the probe and the colony DNA. Washing the filter with a suitable solution helps to remove any unbound probes. Colonies on the film present as dark spots get detected using an autoradiography technique. Shuttle vectors and expression vectors also help in screening the genomic libraries.

Complementation of the mutations:

This test relies on the expression of the wild-type gene. Complementation test determines whether the two mutant sites are in the same gene. Crossing the two mutant phenotypes results in wild-type progeny leading to complementation. Let us consider an example of a yeast mutant lacking the arginine biosynthesis due to a mutant arg1 gene. The wild-type ARG1 gene synthesizes arginine. A designing a yeast-E coli shuttle vector involves the synthesis of the ARG1 gene product. The genomic library containing the ARG1 gene gets transformed into yeast containing the mutant phenotype, the ARG1 wild-type gene gets expressed. Hence, it results in complementation. Therefore, the ARG1 gene overcomes the functional defect of the arg1 mutant gene. The plasmid isolated from the cells helps in the characterization of the cloned gene.

Complementation test helps in identifying genes in the library. Shuttle vectors are bifunctional vectors. They grow and transform into prokaryotes as well as eukaryotes.

Heterologous and oligonucleotide probes:

A small and a defined nucleic acid (either DNA or RNA) identifying the specific molecules with complementary sequences are known as probes. Depending on the type of labeling, the probes matching their complementary sequences get detected through autoradiography. A probe searches a complementary sequence in a cDNA library, a genomic library, northern blot or in situ hybridization. Depending on a good degree of homology between the probes and the genes, the heterologous probes work. The probes consisting of equivalent genes obtained from other organisms are known as heterologous probes. Example, a mouse probe serves as a probe for a human genome library. It involves working with highly conserved genes. Hence, a heterologous probe is similar to the nucleic acid sequence but not complementary to the nucleic acid. Heterologous probing identifies related genes in the same organism. For example, wheat gliadin cDNA clones serve as probes. It hybridizes to a complementary sequence and a variety of other genes. Another example includes a yeast cytochrome c gene as a probe in identifying probable Neurospora cytochrome c clone in a Neurospora gene library.

Oligonucleotide probes identify genes or cDNA libraries. It is possible to clone genes that lack previous genetic information.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Recombinant DNA Principles and Methodologies, James Greene

[3] IGenetics, Peter Russell

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown

© Copyright, 2018 All Rights Reserved.

Plasmid Vectors

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.

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.

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

© Copyright, 2018 All Rights Reserved.

                                                                 

A review on Bacteriophages

Viruses capable of infecting bacteria are known as bacteriophages. These viruses infect the bacterial cells and multiply their genome. They infect various strains of Escherichia coli. The viral properties stop the bacterial replication process. They use the bacterial genetic machinery for their survival and replication. Different institutes worked on bacteriophages and used these viruses as model organisms. It is possible to map the genes with the help of phage genome studies. The bacteriophages follow two types of reproductive cycles such as lytic and lysogenic cycle. Different types of phages exist in the world of microbes.

Bacteriophage consists of a protein coat and a genome. The bacteriophage genetic material consists of DNA or RNA. The bacteriophage structure is made up of capsid head and a filamentous tail. Phage capsid involves proteinaceous coat. The icosahedral capsid is common among bacteriophage. The genome resides in the phage head. During the phage particle assembly, the genetic material gets packaged into the phage head.

There are various classes of bacteriophages. Some of the bacteriophages have a non-enveloped contractile tail whereas some others have an enveloped and rod-shaped tail. Some of them have isometric, ovoid, bottle-shaped, and lemon-shaped structures. It involves variation in the genomes. Some of the bacteriophages have a linear and double-stranded DNA. Examples include T4, Mu, PBSX, and P2 phages. M13 phages have a circular single-stranded DNA. Some other bacteriophages have a circular double-stranded DNA. Linear and segmented RNAs exist in bacteriophages.

Image: Bacteriophage

1.     Bacteriophages as model organisms:

Bacteriophages help in genetic analysis. They serve as vectors in DNA cloning and genetic engineering. It is possible to study genetic recombination using bacteriophages. Bacteriophages infecting bacterial cells produce plaques upon lysis. Hence, it is easy to culture them. Bacteriophages serve as cloning vectors since they exhibit DNA transferring properties.

2. Lytic and lysogenic cycles:

There are two cycles of bacteriophage replication such as lytic and lysogenic cycles. The lytic cycle involves complete lysis of the bacterial cells. The first step involves the infection of bacterial cells. The phage particle lands on the bacterial cell and gets attached to it. Then it interacts with the cell surface receptor and injects its genome into the bacterial cells. Once the phage DNA gets injected into the cell, bacterial replication stops and phage replication process gets initiated. Bacteriophage uses bacterial enzymes for replication. It takes 22 minutes for completing the phage cycle. After completing the replication process, phage particles get assembled and release through the cell. The lysogenic cycle involves the genomic integration into the bacterial chromosome. Depending on the environmental conditions the phage switches over to lytic cycle.

3. Transducing phages:

These phages possess transducing properties. The transducing phages are known as defective phages. During phage genome replication, the defective bacteriophage takes up the bacterial genome. It transfers it to the other bacterium. Hence, the genome gets transferred from the donor bacterium into the recipient bacterium. Two types of transducing phages exist. Generalized transducing phages undergo generalized transduction. Specialized transducing phages undergo specialized transduction. Generalized transduction does not follow any special pattern of transduction.  Any part of the bacterial genome gets transduced. Specialized transduction involves transduction of specific gene or segment of a bacterial chromosome. The defect in the phage becomes an advantage for the bacteria to transfer genes. Transduction does not involve sex pilli of the bacterial cells. It is a phage-mediated process. Bacterial conjugation involves the transfer of F plasmid through sex pill. However, the transduction process directly involves the role of the defective phages.

4. Bacteriophage gene mapping studies:

Studies involving the mutant and wild-type bacteriophage strains helped in mapping the genes. Fine structure and deletion mapping contributed majorly to the field of genome mapping. The idea of overlapping genes came from the bacteriophage gene mapping. Separate mapping of intragenic and intergenic recombinants is possible.

5. Advantages of Bacteriophages:

The bacteriophages are ubiquitous, meaning they exist everywhere. They are highly specific in their action. It is easy to study them using bacterial cultures. Hence, they serve as tools for detecting the pathogenic bacteria. For example, bacteria resistance to specific antibiotics get detected using phage infection. Bacteriophages play a role in therapeutics for tuberculosis. It is possible to improve the TB-vaccine using genetic engineering and bacteriophages.

6. Applications of phages in recombinant DNA technology:

·    Cosmids: A cosmid vector consists of a lambda cos site. Apart from being vectors cosmids serve as probes. They play an important role in FISH and chromosome painting, where cosmid probes get involved. Genomic library preparation using cosmids helps in mapping the genes.

·        For the construction of a cosmid library, we require the phage packaging extract. Here comes the direct role of bacteriophages. The phage packaging extract consists of phage proteins for multiplication, phage lysate, empty phage heads, and unattached phage tails.

·    Phasmids: They are the vectors based on the bacteriophages. A molecular biology technique known as phage display involves the role of these special types of vectors.

·        It is used to study protein-protein interactions and DNA-protein interactions. Phage display involves M13 and filamentous phage.

·   Phage Therapy: Bacteriophages act as anti-bacterial agents since they lyse the bacterial cells. The antibiotic discovery and phages as therapeutic agents still require research and clinical trials, though scientists are working on the same.

·  The role of bacteriophages in the food industry: USFDA approved various bacteriophage products. For example, LMP-102 Intralytix treated ready to eat meat and poultry products. FDA approved LISTEX. It employed bacteriophages for killing the Listeria monocytogenes on the cheese.

·   In vitro diagnostics: The MRSA/MSSA blood test employed bacteriophages for detecting the S aureus and antibiotic-resistant cultures.

·   Contribution in sanitation: Bacteriophages help in sanitation and disinfection of contact surfaces. 
References:
[1] Microbial Genetics, Keya Chaudhari
[2] Molecular Genetics of Bacteria, Jeremy W. Dale, Simon F. Park
[3] Genetics, G. Ivor Hickey

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