In vitro packaging (B.SC. Microbiology Semester VI- RDT)
In vitropackaging
Packaging requires a number of different proteins coded by the lambda genome. These proteins can be prepared at ahigh concentration from cells infected with defective lambda phage strains. Two different systems are in use.
With the single strain system, the defective lambda phage carries a mutation in the cos sites, so that these are not recognized by the endonuclease that normally cleaves the catenates during phage replication. This means that the defective phage genome cannot pack within capsid though it does direct synthesis of all the proteins needed for packaging. The proteins accumulate in the bacterium and can be purified from the cultures of E. coli if infected with the muted lambda. The protein preparation is then used for in vitro packaging of recombinant lambda molecules.
With the second two strain system two defective lambda strains are needed. Both of these strains carry a mutation in a gene for one of the components of the phage protein coat: with one strain the mutation is in gene D and with the second strain it is in gene E. neither strain is able to complete lytic cycle in E. coli because in the absence of the product of the muted gene the complete capsid structure cannot be made. Instead the products of all the other capsid protein producing gene accumulates. The E protein, which is the major component of the phage head; mutants that are defective in this gene are unable to assemble the preheads, and therefore accumulate the other, unassembled components of the phage particle as well as the other proteins involved in packaging. The D proteins, which are involved in the packaging process itself. Mutants that are defective in these genes are able to produce the preheads, but will not package DNA. This results in the accumulation of empty preheads.
During in vitro packaging, a mixture can therefor be prepared by combining lysates of two cultures of cells- one infected with mutant D strain and the other infected with the mutant E strain. The mixture now contains all the necessary components for in vitro packaging (each lysate contains phage tails, assembly proteins and components of the head, except either D or E).
Although mixing is done in vitro the components can self-assemble into a functional phage that can infect E. coli.