Lambda Phage Vectors

Lambda Phage Vectors - Plasmid vectors described in the previous section are often used for cloning DNA segments of small size (upto 10 kilobases). However, while preparing a genomic library in a eukaryote, the cloned fragments should be large enough to contain a whole gene.

This will also allow cloning of the whole genome into a number, which will not be unreasonably large and therefore can be screened without serious difficulty

The above properties and other requirements of cloning whole genome In eukaryotes are fulfilled by the phage lambda and cosmid vectors, the former permitting cloning of segments upto 20-25kb long (kb = kilobases) and latter accommodating segments upto 45kb long. Phage lambda (A).

However, is easier and more efficient for making genomic and cDNA Libraries

(a) λgt10 and λgt11. λgt10 and λgt11 are modified lambda phages designed to clone cDNA fragments. The major difference between these two Vectors is that λgt11 is an expression vector, where inserted DNA is expressed as β galactosidase fusion protein.

λgt10 is a 43 kb double stranded DNA for cloning fragments that are only 7 kb in length. The insertion of DNA inactivates c+ (repressor) gene generating a cl- bacteriophage. Non recombinant λgt10 is cl+ and forms cloudy plaques on appropriate E. coli host, while recombinant cl- λgt10 forms clear plaques permitting screening of recombinant plaque.

Further, in an E. coli strain carrying hf lA 150 mutation (high frequency lysogeny mutation) only cl- phage will form plaques, because cl+ will form lysogens (integrate with bacterial genome) and will not undergo lysis to form any plaques. Recombinant λgt10 plaques can thus be easily selected

λgt11 is a 43.7 kb double stranded A phage for cloning DNA segments, which are less than 6 kb in length (usually for cDNA). Foreign DNA can be expressed as β galactosidase fusion proteins. Recombinant λgt11 can be screened using either nucleic acid or antibody probes (λgt10 can not be screened using antibodies).

The recombinant λgt11 becomes gar, while non recombinant λgt11 remains gal+, so that an appropriate E. coli host, with recombinant phage (gar) will form white or clear colonies and that with non recombinant phage (gal+) will form blue colonies permitting screening in the presence of IPTG (inducer) and Xgal (substrate).