Cosmid Vector Library Construction

 

Cosmid vectors

  • Cosmid are plasmid type of vector (example- c2RB, pJB8)
  • Cosmid vectors are hybrids of plasmid and bacteriophage lambda DNA (a small plasmid containing the plasmid origin of replication (ori), an antibiotic resistance gene such as ampR and a suitable restriction site for cloning along with the COS sequences from phage DNA.
  • Because of the COS sequence, cosmid recombinants can be packaged into viral particles allowing high efficiency transformation but the distance between two cos sites should be 45-50 kb i.e., if the insert size is smaller than this then it can’t go for packaging.
  • Since the recombinant DNA does not encode any lambda proteins, cosmids do not form viral particles (or plaques) but rather forms large circular plasmids and the colonies that arise can be selected on antibiotic plates, like other plasmid DNA recombinants.
  • Advantage of cosmids are that they can insert large DNA compared to plasmid and the transformation efficiency is very high as phage can infect E coli very easily.
  • The main disadvantage of cosmids is their inability to accept more than 40 – 50 kbp of DNA.
  • Some cosmid clones can be unstable during the propagation in E. coli because of the high copy number.

Cosmid Library construction:

Plasmid Vector PDF
cosmid library construction
  • In order to clone a gene of interest into the cosmid, both the gene and cosmid should be cut either with the same restriction enzyme or with two enzymes that will produce identical sticky ends (e.g., BamHI and MboI, as in Fig).
  • Note that, the vector is digested two times in order to create cos sides at both ends along with the open up.
  • Then mixed in the presence of DNA ligase for the formation of recombinant vector.
  • After suitable time interval, the recombinant vectors with DNA fragments, packaged into phages through in vitro packaging method using cos sites of cosmids.
  • Then recombinant vectors are transformed to E.Coli with transduction method, the cos end joins forming a circular DNA.
  • Transformed E.Coli culture then allowed to grow in a suitable selection medium with ampicillin since vector contains ampicillin resistance gene. For this reason, only the recombinant vector containing colonies grew in the medium.

Advantages of using cosmids as vectors:

  • They have high transformation efficiency and they are capable of producing a large number of copies from a small quantity of DNA.
  • Also, they can carry up to 45 kb of insertion compared to 25 kb carried by plasmids and λ.

Disadvantages of using cosmids as vectors:

  • Cosmids cannot carry more than 50 kb of the insert.

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