Bacteriophages as vectors
Bacteriophages provide another source of cloning vectors. Since usually, a phage has a linear DNA molecule, a single break will generate two fragments, which are later joined together with foreign DNA to generate a chimeric phage particle (Fig. 39.9). The chimeric phage can be isolated after a lytic cycle. The use of phage particles as vector imposes a limitation on the size of foreign DNA. which can be cloned, because the capacity of phage head is only limited, and if the size of foreign DNA is too long, size of phage DNA may not be accommodated in phage head. In order to overcome this problem, those segments of DNA, which do not contain essential genes may be removed. Such a technique has been followed in phage lambda (λ)to create a smaller vector genome having single restriction site for the enzyme EcoRl. Since the reduced size also fails to be adequately packed in phage head (there is also a requirement of a minimum size of DNA), this automatically provides a selection method, in which only the chimeric particles will be obtained in the phage progeny, and vector particles lacking cloned segment will be eliminated due to its reduced size.
|Fig. 39.9. Cloning of foreign DNA in a nonessential region of phage vector.
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 (of clones), which will not be unreasonably large and therefore can be screened without serious difficulty. These properties and other requirements of cloning whole genome in eukaryotes are fulfilled by the phage lambda, cosmid or phagemid vectors, the former permitting cloning of segments upto 20-25kb long (kb = kilobases) and latter accommodating segments upto 45kb long. Phage lambda (k
however is easier and more efficient for making genomic and cDNA libraries. Some of the vectors derived from lambda and M13 phages include the following :
λgt10 and λgt11. λ
gt10 is 43kb double stranded DNA for cloning fragments that are only upto 7kb in length. The insertion of DNA inactivates c/+
(repressor) gene generating a cI-
bacteriophage. Nonrecombinant λ
gt10 is cI+
and forms a cloudy plaque on appropriate E. coli
host, while recombinant cI- λ
gt10 forms clear plaques permitting screening of recombinant plaques (see Regulation of Gene Expression 2. Cricuit of Lytic Cycle and Lysogeny in Bacteriophages
). Recombinant λ
gt10 plaques can thus be easily selected.
gt11 is a 43.7kb double stranded λphage for cloning DNA segments, which are less than 6kb in length (usually for cDNA). It differs from λ
gt10 in being an expression vector so that foreign DNA can be expressed as β galactosidase fusion proteins. The recombinant λ
gt11 becomes gat,
while nonrecombinant λ
gt11 remains gal+,
so that on an appropriate E. coli
host, in the presence of IPTG (inducer) and Xgal (substrate), recombinant phage (gal-
)will form white or clear plaques and non-recombinant phage (gal+
)will form blue colonies permitting screening.
EMBL3 and EMBL4.
In these vectors a central non-essential port of 44kb long phage can be replaced by a foreign DNA which may be as long as 20-23 kb. Therefore, they are called replacement vectors. Non recombinant DNA is too small for packaging, so that there is automatic selection against non-recombinant phages (Fig. 39.10).
Charon 34 and Charon 35.
|Fig. 39.10. Use of lambda (λ) phage as vector for cloning eukaryotic DNA fragments.
These vectors will accept fragments 9-20kb (kilobases) long.
M13 as cloning vector for DNA sequencing.
M13 is a filamentous bacteriophage of E. coli
and contains a 7.2kb long single stranded circular DNA. M13 phage has been variously modified to give rise to a MP13 mp series of cloning vectors which can be used for cloning of a wide variety of DNA fragments particularly for the purpose of sequencing through Sanger's method of dideoxy chain termination (for details of sequencing methods, consult next main topic).
Cosmids and phagemids.
Cosmids are plasmid particles, into which certain specific DNA sequences, namely those for cos
sites are inserted. Since these cos
sites enable the DNA to get packed in lambda particle, cosmids allow the packaging of DNA in phage particle in vitro,
thus permitting their purification. Like plasmids, these cosmids perpetuate in bacteria and do not carry the genes for lytic development.
Phageminds are prepared artificially by combining features of phages with plasmids, as the name suggests. One such phagemid, which is commonly used inmolecularbiology laboratories is pBluescript II KS,
which is derived from pUC19, and is 2961 base pairs long. This is an expression vector, since the cloning site is flanked by T3 and T7 promoters to be read in opposite directions.