Bacteriophages As Cloning Vectors PDF

Bacteriophag es As Cloning Vectors  Cloning of a large fragment of DNA in a plasmid is not easy. Artificial genetic transformation of a large Advantages plasmid to E. coli cells is very difficult and inefficient. of phage cloning...

Bacteriophag es As Cloning Vectors  Cloning of a large fragment of DNA in a plasmid is not easy. Artificial genetic transformation of a large Advantages plasmid to E. coli cells is very difficult and inefficient. of phage cloning over - DNA can be packed in vitro into phage plasmids particles and and transduced into E-coli with high efficiency - Foreign DNA upto 25Kb in length can be inserted - Screening and storage of recombinant DNA. is easier  Bacteriophages such as lambda have been modified to make useful cloning vectors. Bacteriophage 1) It can hold larger amounts of DNA than plasmids (20 kb) Lambda as a Cloning Vector 2) DNA can be efficiently packed into phage particles in vitro and infect E. coli In the construction of genomic library, it is often helpful to maintain larger pieces of DNA. Types of phages The lambda phage A phage (from 'bacteria' and Greek phagein (fagein), 'to eat') is a virus that infects and sometimes lyses bacteria. Phages can be found in many reservoirs populated by bacteria such as soil, the intestine of animals etc. One of the densest natural sources for phages is seawater, where up to 2.5x108 virions per milliliter have been found. Lysis and Lysogeny The lambda phage is adorbed on the lamb receptor. This receptor is only expressed when the bacteria are grown in the presence of maltose. Such a bacteriuml is known as “competent”. Lysis: multiplication. Most phages take over the machinery of the host cell to produce a large number of viral particles. This result in the lysis of the host cell, which releases the newborn phages. 2. Lysogeny: rest. In some infections, a phage stays silent within the cell. This silent phage can go lytic under certain conditions. a lysogenic phage is called a prophage (integrated into the chromosome) Lysogenic bacteria have immunity against further infection The prophage can be induced and is excised from the bacterial genome  Who is controlling the process? Lysis and lysogeny are controlled by the proteins encoded by cro and cI genes. The lambda phage will remain in the lysogenic state if cI proteins predominate, but will be transformed into the lytic cycle if Cro proteins predominate cI genes transcription and translation regulate lysis/lysogeny Lytic or Virulent Phages What controls whether a bacteriophage Lambda represser : lambda will take the lytic or the lysogenic a protein encoded by route? the gene cI gene and it prevents the For lysogeny synthesis of all other proteins 1) The production of all proteins must be prevented 2) A copy of the lambda genome must be integrated into the host chromosome  One of the best-studied temperate phages, which infects E-coli Lytic and lysogenic cycles present Tailed bacteriophage- no tail fibers are present Lambda bacteriophages Double stranded DNA. At the 5‘ terminus of each strand is a single stranded ends (12bp)- CGGGCGGGCGACCTCG They are complementary and when the two ends are free in the host they associate (the cos site)  Insertion vector - have unique cleavage site (single) λ –based - small piece of foreign DNA is inserted cloning - foreign DNA does not affect the function of phage vectors - minimum size of the vector must be above 35Kb and maximum size of the insert is about 18Kb (maximum total size 53Kb)  Replacement vector They have cleavage sites present on either site of a non-essential DNA of phage λ –based Each terminal site after cleavage will have a cos site cloning - 25-30 kb of the phage genome codes vectors for essential products for the lytic cycle, the remaining 20-25kb could be replaced