Cosmid Vectors

In vitro packaging using lambda lysates is a powerful technique. Packaging of DNA into a lambda head does not require the lambda genes, if fact, it is possible to fill almost in vitro packaging Procedure in which virus proteins are mixed with DNA in vitro to assemble infectious virus particles. Often used for packaging recombinant DNA into bacteriophage lambda

Induce l LYSIS

Tails

Assembly proteins Protein E

Pre-heads (due to lack of D)

Lambda DNA with insert

Lambda DNA with insert

Successful packaging

Tails

Assembly proteins Protein D

No pre-heads (due to lack of E)

FIGURE 22.20 In Vitro Packaging of Lambda Replacement Vector

A lambda cloning vector containing cloned DNA must be packaged in a phage head before it can infect E. coli. Before the DNA can be packaged, the phage head proteins must be isolated. To do this, a culture of E. coli, is infected with a mutant lambda which lacks the gene for one of the head proteins called E. A different culture of E. coli is infected with a different lambda mutant, which lacks phage head protein D. Both E. coli cultures are grown with the mutant lambdas and the viruses are induced to enter the lytic cycle. Although the E. coli are lysed by the phage, they cannot form complete heads. Instead a soluble mixture of phage proteins is isolated. Each lysate contains phage tails, assembly proteins, and components of the heads, except either D or E. These two lysates are mixed along with the lambda vector containing the cloned DNA. Although mixing is done in vitro, the components can self-assemble into a functional phage that can infect E. coli.

the whole of a lambda particle with cloned DNA by using cosmid vectors. Cosmids themselves are small multicopy plasmids that carry cos sites (Fig. 22.21). The cosmid is first linearized so that each end has a cos sequence. In order to clone a gene of interest into the cosmid, both the gene and cosmid are cut either with the same restriction enzyme or with two enzymes that give identical sticky ends (e.g., BamHI and MboI, as in Fig. 22.21). The target DNA is often only partially digested, i.e., some sites are left uncut. First, this allows large segments of a genome to be isolated. Second, if a cut cosmid Small multicopy plasmid that carries lambda cos sites and can carry around 45 kb of cloned DNA

FIGURE 22.21 Cosmid Vector

To clone large pieces of DNA into cosmid vectors, both must have compatible sticky ends. The cosmid vector is first linearized so that each end has a cos site. Then the linear cosmid is cut with BamHI, which generates sticky ends with the overhang sequence GATC. The genomic DNA from the source of interest is also digested. Instead of BamHI, this DNA is partially digested with Mbol, which also generates a GATC overhang. Partial digestion leaves some sites uncut and allows large segments of a genome to be isolated. These segments are mixed with the two halves of the cosmid and joined using ligase. The final constructs are packaged into lambda particles in vitro and are used to infect E. coli.

BamHI

Linearize

Seal

AmpR

Open

BamHI

1) PARTIAL Mbol DIGEST 2) Mbol FRACTIONATE BY SIZE

DNA fragments of approximately 40 kb i/jrvr'tr

/fisrvr tfwwwwt

BamHI

t4 DNA LIGASE

cos J^wvwwwwwwvwwi ori cos

In vitro packaging

site lies within a gene of interest, some fragments will still carry the intact gene. Liga-tion of the two cosmid pieces to either side of the target DNA results in a length of DNA with a cos site at each end. This construct can be packaged into lambda particles in vitro, and then used to infect E. coli. Using a small cosmid, of say 4 kb, allows inserts of up to about 45 kb to be cloned.

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