Question

Once you had obtained a highly concentrated stock of purified phage, how would you separate the recombinant phage
DNA away from the phage head and tail proteins?

Answer 1

Answer;

Phage ;A bacteriophage (/bækˈtɪərioʊfeɪdʒ/), also known informally as a phage (/feɪdʒ/), is a virus that infects and replicates within bacteria and archaea.

The term was derived from "bacteria" and the Greek φαγεῖν (phagein), meaning "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes (e.g. MS2) and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm.

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Recombinant DNAt depends on the ability to produce large numbers of identical DNA molecules (clones). Clones are typically generated by placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell. When a single vector containing a single DNA fragment is introduced into a host cell, large numbers of this fragment are reproduced along with the vector.
Once the genomic DNA is isolated and purified, it is digested with restriction endonucleases.
These enzymes are the key to molecular cloning because of the specificity they have for particular DNA sequences. It is important to note that every copy of a given DNA molecule from a specific organism will give the same set of fragments when digested with a specific enzyme. By digesting complex genomic DNA from an organism it is possible to reproducibly divide its genome into large number of small fragments, each approximately the size of a
single gene. Some enzymes cut straight across the DNA to give flush ends or blunt ends.
Other restriction enzymes make staggered single strand cuts, producing short single stranded projections at each end of the digested DNA. These ends are not identical but complementary and will base pair with each other, they are therefore known as cohesive or sticky ends. In addition the 5’ end projections of the DNA will always retain the phosphate group.

?DNA and cDNA cloning
A number of phage vectors are used in DNA and cDNA cloning. Perhaps the most widely used are phage lambda vectors, which can be used both for cloning of mammalian DNA fragments (genomic DNA libraries) and for the cloning and expression of mammalian cDNAs(cDNA libraries). The overall cloning strategy is illustrated on the next page. Briefly, genomic DNA is fragmented into pieces of about 15-20 kb in size and then ligated to the "arms" of a predigested lambda phage cloning vector. This results in the production of concatenated molecules which are cleaved and packaged into phage heads using commercially available packaging reactions. The packaged phage particles are used to infect susceptible E. coli strains (strains are usually grown in the presence of maltose, since this induces expression of the bacterial lamB gene, which transports maltose into the cell and serves as the receptor for lambda phage). The phage plaques which result from infection of susceptible bacteria can then be screened with radioactive DNA probes or with antibodies, to detect the desired DNA fragment or the protein product of the desired cDNA clone.

Important considerations in the use of phage lambda as a cloning vehicle include the following:
1. The vector systems are of the genereplacement type. That is, the final recombinant clones remain infectious for E. coli since the
genes which are deleted from the phage are non-essential essential for lytic phage growth. Thus, typical lambda phage vectors are in many ways analogous to certain mammalian virus vectors - such as vaccinia virus or herpesvirus vectors in which a foreign gene has replaced some non-essential viral gene (such as the gene encoding
thymidine kinase).
2. The phage's genome cleavage and packaging machinery makes specific nucleolytic cleavages at the cohesive ends between concatemeric genomes (so called cos sites). This releases the genome unit-length molecules for packaging. Many mammalian viruses also have specific terminal sequences which are important for genome replication and packaging.
3. The wild-type phage genome (i.e., 38-53 kb or so), since the lambda phage head has a tight constraint on the amount of DNA that it will accomodate. The packaging size limitation of lambda phage vectors is one which is common to most mammalian
virus vectors also (i.e., recombinant viral genomes must usually be of wild-type length + 5% or so). Two of the few exceptions are the bacteriophage M13 and mammalian rhabdoviruses (such as vesicular stomatitis virus), both of which can accomodate genomes considerably (>10%) larger than wild-type. This is probably
because of the rod-like structure of these viruses (bullet-shape in the case of rhabdoviruses); an increase in the length of the rod allows the particles of these viruses to accomodate an extended genome.Colony OR Plaque Hybridization for Screening of Libraries
Once a genomic library or cDNA library is available, we may like to use it for isolation

bacteriophage particles is essential for rational design of bacteriophages with defined pharmacokinetic parameters and to identify the mechanisms of immunobiological activities demonstrated for some bacteriophages. This work requires highly purified preparations of the individual phage structural proteins, possessing native conformation that is essential for their reactivity, and free of incompatible biologically active substances such as bacterial lipopolysaccharide (LPS). In this study we describe expression in E. coli and purification of four proteins forming the surface of the bacteriophage T4 head: gp23, gp24, gphoc and gpsoc. We optimized protein expression using a set of chaperones for effective production of soluble proteins in their native conformations. The assistance of chaperones was critical for production of soluble gp23 (chaperone gp31 of T4 phage) and of gpsoc (chaperone TF of E. coli). Phage head proteins were purified in native conditions by affinity chromatography and size-exclusion chromatography. Two-step LPS removal allowed immunological purity grade with the average endotoxin activity less than 1 unit per ml of protein preparation. The secondary structure and stability of the proteins were studied using circular dichroism (CD) spectrometry, which confirmed that highly purified proteins preserve their native conformations. In increasing concentration of a denaturant (guanidine hydrochloride), protein stability was proved to increase as follows: gpsoc, gp23, gphoc. The denaturation profile of gp24 protein showed independent domain unfolding with the most stable larger domain. The native purified recombinant phage proteins obtained in this work were shown to be suitable for immunological experiments in vivo and in vitro.