4.1 Bacteriophage Lambda Vectors
Interestingly enough, even bacteria are plagued by viruses! You may know that when viruses infect cells they manage to transfer their nucleic acid genetic material to the inside of the host cell. There it replicates to produce up to several hundred new viruses. If we could replace one or more of the viral genes with a gene we are interested in amplifying, then we would have another type of vector in addition to the plasmids alreaady discussed. Furthermore, the host cell may obligingly package up the replicated DNA containing our gene into new virus particles that are liberated from the cell.
The lambda genome is 48.5 kbases in length and contains about 46 genes.
The bacteriophage lambda is frequently used as a vector. It has a head structure
that contains the viral DNA and a long tail which is used in attachment
of the virus to E. coli. The original virus has a set of genes that
allows the viral DNA to insert itself into the bacterial chromosome. These
genes may be cut out and discarded to provide a location for our new gene
to be inserted.
Figure 28. The lambda genome
Following attachment of the virus to E. coli, the DNA in the head structure travels down the hollow tail and enters the host cell. The linear, viral DNA has naturally "sticky ends" consisting of a complementary sequence of 12 bases on each end of the molecule. The sticky ends of the linear DNA (the cos site) associate to form circular DNA. The virus codes for its own DNA polymerase which leads to the synthesis of several hundred copies of viral DNA.
Figure 29. The phage life cycle
The DNA is synthesized in very long repeated units which are cleaved at the cos sites to give unit length viral DNA for insertion into new particles. The bacteriophage DNA also codes for all of the proteins needed to put together new virus particles which then are assembled inside the host cell.
The new DNA is trapped inside the newly-formed viral coat proteins and the host cell bursts, liberating the new particles. A most remarkable property of the proteins and the viral DNA is that complete particles will assemble in the test tube when all of the reactants are mixed together.
The infection spreads to adjacent cells and the cycle is repeated . If the infection is carried out with E. coli cells that are spread over an agar surface, the infected areas will appear as clear dots (or plaques) surrounded by live cells. Bacteriophage may be recovered from the cear areas. Each plaque originates from a single infected cell.
Figure 30. Phage lambda plaques on a lawn of bacteria
Virus particles are separated from cell debris and the DNA isolated. The process may be scaled up by infecting cells growing in liquid culture. Over 100 different lambda vectors have been prepared for use in cloning. The DNA of the cloning vector must be the correct size for it to be packaged into virus particles (between 38 and 51kb).
The vector lambda gt10 (43.8 kbases) has an EcoRI site inserted within the cI gene (lambda repressor) and so could accept a DNA fragment of 7.6 kbases before becoming too large to be packaged into virus particles.
Figure 31. The vectors lambda gt10 and Charon 16A
Other lambda vectors can incorporate DNA fragments of up to 22 kbases (e.g., the Charon series).
To use lambda gt10, we would cut the vector DNA and the DNA to be inserted with EcoRI, incubate the two together, seal with ligase and incorporate the recombinant molecule into particles in the test tube: