Lambda Vectors
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Vectors
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Vector Systems |
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Biology |
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Vectors |
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The analysis of lambda transducing phage showed that approximately 40% of the wild-type genome is not required for replication by the lytic life cycle. This 40% can therefore be replaced with foreign DNA without affecting the ability of the phage to form plaques. However, although 40% of the lambda genome is dispensible, virus particles containing less than 75% (and more than 105%) of the wild-type genome length (50KB) spontaneously fall apart. Therefore, viral genome packaging imposes a size constraint on the use of lambda as a cloning vector.
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Lambda Insertion Vectors
Insertion vectors are the simplest form of lambda cloning vectors. In the construction of insertion vectors, 25% of the dispensible portion of the wild-type lambda genome was eliminated. Insertion vectors must also contain a unique restriction site into which we can insert foreign DNA fragments. Since the lambda genome approximates a random 50 KB DNA sequence, we can expect most 6-mer restriction enzymes to cut several times (average frequency is 1/4000 bp). Removal of unwanted restriction sites involved mutagenesis of phage followed by selection for efficient growth on hosts carrying different restriction/modification systems. After multiple rounds of mutagenesis and selection, phage genomes were recovered that lacked unwanted restriction sites. Since insertion vectors are large enough to be packaged into viable viruses, it is useful to have a the lambda cI gene product - the lambda repressor. The lambda repressor acts to shut off lambda transcription. As noted previously, E. coli containing a lambda provirus (a lambda lysogen) are immune to subsequent phage infection and so can grow in the presence of the virus. While this visual morphology difference allows us to identify recombinant and non-recombinant plaques, it would be nice to have a selection system that allowed only recombinant phage to grow.
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The limitation to using lambda insertion vectors is their small insert size capacity. The analysis of transducing phage suggested that as much as 40% of the wild-type genome is dispensible for lytic growth. However viral genomes only 60% of the wild-type length aren't packaged into viable phage particles. Therefore, in order to utilize the full carrying capacity of the lambda vector, substitution phage vectors were developed. In order to maintain the vector as a viral stock it must replicate efficiently. To take advantage of the full carrying capacity (vector constitutes 60 % of recombinant genome length, the insert 40 - 45%), Substitution vectors (10 - 20 KB) carry larger inserts than insertion vectors (0 - 10 KB). Selection is less of an issue with substitution vectors. The removal of the stuffer fragment leaves a viral genome that cannot be efficiently packaged, so non-recombinant phage are (theoretically at least) inviable. Of course, it is difficult to completely remove the stuffer fragment.
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The selection system used to selectively replicate recombinant phage involves yet another observation made by phage geneticists.
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