During the development of new high-capacity lambda vectors, all of the nonessential genes were "thrown overboard." Taking this concept to an extreme, the ultimate goal is to have plasmid-like elements that can be introduced into cells by transduction or conjugation, rather than transformation.
Bacterial artificial chromosomes
We have had an opportunity to learn about several methods by which DNA is taken up naturally by E. coli:
There is one more method that we should discuss, called "conjugation." To some extent, we have already touched on the matter in our previous discussion of the sex factor F in bacteria. As you may recall, the filamentous bacteriophage only infected male bacteria - i.e. those with pili on their surfaces. What causes a bacterium to be male or female? It is the presence or absence of the F factor (integrated or in plasmid form) that determines the sex. Conjugation is the process by which a cellular bridge is formed between two cells (one of which is male), and a single stranded DNA molecule is transferred from one to the other. If the F factor plasmid is transferred, the recipient becomes a "male" bacterium.
This was discovered in 1946 by Lederberg and Tatum, about 18 years after transformation was first described by Griffith. I suppose if F factor transfer had been discovered in the 1990's we would have called these "transsexual" or "transgender" bacteria, but the 1940's were a much simpler time!
The F factor plasmid is nicked at its origin and replicates as a rolling circle, causing a single-stranded DNA to be produced. It takes a bit of time for the entire F factor to be replicated, and if the bacteria are interrupted during the act, only the DNA that has made it through the cellular bridge will be transferred.
In cases where the F factor resides in the genome as an integrated copy (Hfr strain, where Hfr = high frequency recombination), then the rolling circle is the entire E. coli chromosome!
Genes replicated first from the F factor origin (i.e. those on the 3' side of the origin of replication) are more likely to be transferred to the new host because the conjugation only needs to be maintained for a few seconds. On the other hand, genes replicated last on the rolling circle are less likely to be transferred. In this way, it was possible to construct a genetic map of the chromosome, through purposeful interruption of the conjugating bacteria after specific time intervals, and then determining which genes were transferred at high probability. The conjugating bacteria are interrupted during the act (in the Wollman and Jacob experiment) by putting them into a blender and turning it on to "frappe". For the coupling bacteria, that's the equivalent of turning a hose on "those dogs in the yard" .
If the F factor is excised from an Hfr strain abnormally, a new plasmid is generated that may contain novel sequences. We call this an F' factor, to distinguish it from the previously described "F factor", which has a precise meaning. Many times in molecular biology we use bacterial strains that have particular genotypes involving F' factors. The cells that we use in transformation are:
What a mess! You will notice a few old friends in this genotype, however. The indication is that an overexpressing allele of the lac repressor (lacIq) is present on an F' plasmid. What else is there? A transposable element called Tn10, carrying a tetracycline resistance marker. If you grow Top10F' cells on tetracycline containing medium, you can make sure they will keep the F' plasmid carrying the lacIq gene. The lacZdeltaM15 is the C-terminal portion of lacZ that, in combination with lacZ-alpha (the N-terminal portion) in trans, can function as a beta-galactosidase enzyme. We need that gene in order to be able to use blue-white screening with some of the common cloning plasmids we've discussed. The lacZdeltaM15 was introduced into the strain by transduction, on a phage species called phi80.
Now that we've learned a bit about F factors, you might imagine how a cloning vector could be created that was based on an F factor origin of replication. We call such engineered F' plasmids "BACs" or Bacterial Artificial Chromosomes. BACs are capable of carrying approximately 200 kbp of inserted DNA sequence, and the F factor origin of replication maintains their level at approximately one copy per cell. Of course, we needn't stop there! We can also use "YACs" which are Yeast Artificial Chromosomes, and depend on being able to replicate and be maintained in Saccharomyces cerevisiae. YACs can carry approximately 500 kbp of foreign DNA, though they are often criticized due to the problem of natural recombination in the host.
Handling DNA of this size is a real problem, as I have mentioned before, due to the potential for shearing. The way this is solved is to embed the cells from which a library is going to be made, in low melting point agarose. The cells can be lysed in the agarose, simply be incubating the blocks of agarose in sodium dodecyl sulfate (SDS), proteinase K and EDTA. Once the lysis buffer has been washed away, the DNA in the blocks can be digested with a restriction enzyme. When you're ready to ligate the DNA into a F1-based vector, you incubate the block with an enzyme called agarase which digests the agarose matrix. The ligated DNA is then introduced into E. coli by transformation, using electroporation (electric shock) methods to achieve high efficiency.
Here is an example of a BAC vector, from those folks in Buffalo:
In looking at the BAC vector from RPCI, you should notice a couple of important elements:
How do you go about using PACs and BACs in your research (the easy way)?
Obtain a gridded membrane containing spots of BAC/PAC clones. This is analogous to the "bacterial colony" lift that we discussed, except that the spots are organized and numbered.
Department of Biology
California State University Northridge
Northridge CA 91330-8303
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