Structural Biochemistry/DNA recombinant techniques/Plasmid/Lytic Pathway
During the lytic pathway, a lambda bacteriophage containing viral DNA attaches to a bacterial cell. The virus DNA is then inserted into the host cell containing plasmid DNA. The viral DNA from the lambda phage is not introduced into the plasmid but remains in the cell. The viral DNA uses the host cells cell machinery in the process of quickly replicating the viral DNA. The replicated viral DNA is then packaged into virus particles made from viral proteins. Then the virus uses the host cell's machinery and metabolism in order to drive the packaging of the viral DNA into protein coat encasements. After the progeny lambda DNA is replicated and encased, the host bacterium lyses or bursts and releases the numerous copies of the lambda phage to repeat the process on other host cells.
Lamda phage in Cloning
A lambda phage is also a vector used for DNA cloning. It is a virus containing double-stranded phage DNA that can is introduced into E. Coli for replication. Mutant lambda phages have been created that makes cloning easier. One example is the lambda-gt-lambda-beta phage. It contains two EcoRI cleaves sites. The middle section of the phage DNA is removed by restriction digestion. The two remaining arms of the DNA cannot be packaged in to lambda virion because it is too small. Instead, a foreign piece of target DNA is joined to the two arms with ligase, which replaces the middle section that was removed. The addition of the foreign DNA allows the phage DNA to be long enough to be packed into a virion. The packaged lambda virion can then be inserted into a host cell and then replaced through the lytic or lysogenic pathway.
Genomic Library refers to the complete set of thousands of recombinant-plasmid clones, each carrying copies of a particular segment from the initial genome. A researcher can save a library as such and use as a source of gene interest or for genome mapping. A genomic library made using phage is stored is collections of phage clones Whatever cloning vector, the restrictior enzymes do not respect gne boundaries in cutting up genomic DNA, therefore some gene in a genomic library can be divided into two or more clones.
Lambda phages can also be used to house a genomic library. First, a genomic DNA is fragmented by enzymatic digestion. The needed fragments are about 15 kilobases long, which can be separated by gel electrophoresis. Synthetic linkers are attached to the ends of the fragments. The fragments are then introduced into the mutant phage DNA which are then packaged into virions. The virions are introduced to E. coli hosts which then produce many copies of the phage DNA. The progeny virions then contain a genomic library of a DNA that has been fragmented. The genomic library can be screened to isolate a specific gene of interest.
Complementary DNA (cDNA)
Researchers are able to make even more limited kinds of gene library using complementary DNA (cDNA). Once they are able to isolate mRNA from cells, they can actually onatin a mixture of all the mRNA molecules in the cell that have transcribed from a number of different genes. Therefore, the cDNA that is made is a library of a collection of genes. cDNA represents only a part of a cell's genome- the genes that were transcribes in the starting cells.
This kind of genomic library has its advantages, for example if a researcher wants to study the genes responsible for a specialized function of a particular kind of cell, such as the liver or brain cell, the smaller more specific library allows this to be done with greater detail. Also, by making cDNA from cells of the same type of an organism at different times, a researcher can trace changes that occur in gene expression.
There are a total of six stages in the Lytic Lifecycle of the Lytic Bateriophage. The six stages are Adsorption, Penetration, Replication, Maturation, Release and Reinfection.
Stage I: Adsorption
In the first stage of the lytic bateriophage life cycle, the attachment sites on a phage is abosrbed on the receptor site of the host cell. The phage usually attach itself to the cell-wall fo the cell, sometimes it can attach to the flagella or pilli of a bacteria as well.
Stage II: Penetration
In the second stage, the phage has a specific enzyme that makes a hole in the cell wall and then inserts the phage's genome into the cell's cytoplasm. The alternative way to insert the genome into the cell is to make a hollow tube that goes into the bacterium. This is called "contracting a sheath".
Stage III: Replication
In the third stage, after the insertion of the phage's genome into the baterium cell, the cell consequently shuts down the synthesis of RNA, DNA and protein. At the same time, the phage replicates its own genome by using the cell's metabloic components to make phage enzymes.
Stage IV: Maturation
In the maturation stage, the genome slows generates itself around the genome to prepare for the next stage.
Stage V: Release
The release stage occurs when the lysozymes from the phage genome breaks down the peptidoglycan. This causes a osmotic lysis of the cell, and the bateriophage is release to the outside of the cell.
Stage VI: Refection
About 50 - 200 phages may be produced from this infected bacterium. Thus after the release of the bacteriophages, they can go and infect other baterium cells near the surroundings.
A specialized labmda transducing phage, containing the rec A gene had been identified in the early 1960s as an essential for homologous recombination in E.coli cells but the product had never been isolated. Robert Lehman and his team decided to embark on this experiment. The rec A protein had ATPase activity dependent on single stranded DNA meaning that it was dependent on DNA ATPase. Rec A protein can promote renaturation, the opposite of denaturation, of the single strand that is complementary but it can also promote ATP dependent strand exchange between single strands and a homologous DNA dublex. It can also form Holiday junctions, a the key factor in recombination where DNA is switched around to form variety. Lehman, Robert. Wanderings of a DNA Enzymologist: From DNA Polymerase to Viral Latency. Annu. Rev. Biochem. January 16, 2006.