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Home >> Biotechnology and Genomics >> Cloning and Expression Vectors >> Insertion Lamda Vectors


Insertion  vectors.

(i) A.gt10 and A.gt11. gt10 and gt11 are modified lambda phages designed to clone cDNA fragments. The major difference between these two vectors is that gt11 is an expression vector, where inserted DNA is expressed as  galactosidase fusion protein.

Insertion lambda Vectors

Insertion lambda vectors lambda gt10 and lambda gt11 and their essential elements


gt10 is a 43kb double stranded DNA for cloning fragments that are only 7kb in length. The insertion of DNA inactivates cI+ (repressor) gene generating a cI- bacteriophage (cI- phage is unable to lysogenize).

Non-recombinant gt10 is cI and forms cloudy plaques due to lysogeny on appropriate E. coli host, while recombinant cI gt10 forms clear plaques due to lysis permitting screening of recombinant plaques. Further, in an E. coli strain carrying hflA150 mutation (high frequency. lysogeny mutation) only cI phage will form plaques, because cI will form lysogens (integrate with bacterial genome) and will not undergo lysis to form any plaques. Recombinant gt10 plaques can thus be easily selected.

Non-recombinant gt10 is cI and forms cloudy plaques due to lysogeny on appropriate E. coli host, while recombinant cI gt10 forms clear plaques due to lysis permitting screening of recombinant plaques. Further, in an E. coli strain carrying hflA150 mutation (high frequency. lysogeny mutation) only cI phage will form plaques, because cI will form lysogens (integrate with bacterial genome) and will not undergo lysis to form any plaques. Recombinant gt10 plaques can thus be easily selected.

gt11 is a 43.7kb double stranded  phage for cloning DNA segments, which are less than 6kb in length (usually for cDNA). Foreign DNA can be expressed as  galactosidase fusion proteins. Recombinant gt11 can be screened using either nucleic acid or antibody probes (gt10 can not be screened using antibodies).

The recombinant gt11 becomes gal-, while non-recombinant gt11 remains gal+, so that an appropriate E. coli host, with recombinant phage (gal-) 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).

(ii) Lambda () Zap vectors. These vectors (designed by Stratagene) combine the high efficiency for the construction of recombinant lambda and the convenience of a plasmid system with blue-white colour selection. ZAP vectors also contain complete sequence of phagemid vector pBluescript, which can be excised in vivo.

Lambda Zap Vector

Lambda Zap vector and its characteristic features


These vectors can accommodate DNA inserts, upto 10kb in length and have the following additional features : (i) they can be screened with either the DNA probes or antibody probes; (note: selection and screening are two steps in the construction and use of genomic libraries; selection allows identification of colonies or plaques with recombinant vector molecules; screening allows identification of specific recombinant vector of one's own choice); (ii) they allow in vivo excision of the pBluescript phagemid, thus permitting characterization of the insert in a plasmid system.


Excision of Plasmid pBluescript with the insert from λ Zap Vector

Excision of Plasmid pBluescript with the insert from lambda Zap Vector
Excision of Plasmid pBluescript with the insert from lambda Zap Vector
Excision of Plasmid pBluescript with the insert from lambda Zap Vector

pBluescript also has the becteriophage f1 (single stranded DNA phage) origin of replication (initiation and termination sites) permitting rescue of single stranded DNA, which can be used for DNA sequencing or site directed mutagenesis (exonuclease III and mungbean nuclease can be used for creating deletions at either end); (iii) the polylinker of pBluescript present in ZAP vector provides a multiple cloning site (MCS), flanked by T3 and T7 promoters (these can be used to produce riboprobes or for PCR reactions), and a number of primer sites (T3, T7, M13 forward, M13 reverse, SK, KS) for DNA sequencing. (iv) they also have lacZ promoter to allow expression of fusion proteins (screened through antibodies) suitable for Western blots or protein purification.

As mentioned above, -ZAP vector has been designed to allow in vitro excision and recircularization of pBluescript phage mid carrying the insert. The fl phage region of pBluescript contains the origin of replication (initiator or I and terminator or T) which responds to f1 phage proteins synthesized under the influence of a helper phage (fl).

Therefore, for in vivo excision of pBluescript, E. coli cells are simultaneously infected by lambda vector and f1 helper page. The f1 phage proteins nick one of the two DNA strands at initiator site and allow replication of DNA downstream (upto the terminator T site), thus duplicating complete pBluescript carrying the insert.

The single stranded DNA thus synthesized is circularized by gene II product of fl phage, so that a circular - DNA molecule containing all sequences between initiator and terminator sites is produced (this will include entire pBluescript with the insert, if any; it will not include sequences of lambda phage located outside; Circularization also recreates f1 origin.

Signals for packaging of fl phage are found at the terminator (T) site, so that the circularized and "packaged" DNA is secreted from E. coli. Once phagemid is released, the E. coli cells are destroyed by heating to 70°C (phagemid is resistant to 70°C). For production of double-stranded DNA, the packaged DNA is mixed with fresh E. coli cells (often a different strain) and transformed cells selected on LB-ampicillin plates.

These selected colonies can be used for isolation and analysis of double standred insert DNA for a variety of purposes including DNA sequencing.

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