Temperate Bacteriophage Methods
Transduction as art.
A genetic switch
Let us remember the split life cycle of the temperate bacteriophage lambda:
As we learned in the previous lecture, the nature of immunity was discovered through the isolation of "clear mutants" of lambda that were unable to enter a lysogenic state. These mutated phage were completely virulent and only able to cause a lytic infection. Thus the name "clear" to describe the characteristics of their plaques on a plate. Three loci in the lambda genome could harbor mutations leading to a "clear" phenotype, and these were named cI, cII, and cIII. Infection of a lysogenic bacterium with clear mutants of lambda did not lead to lysis, however! E. coli lysogenic for lambda was immune even to clear mutants.
As shown in the diagram above, lambda DNA is initially transcribed from the promoters PL and PR, which direct synthesis of RNA in opposite directions (left and right respectively). Transcription is initially terminated at sites tL and tR, but expression of the N gene (in green) leads to "antitermination" and production of longer transcripts.
Here's where the idea of a "switch" comes into play ...
The control of the region between the cI gene and cro genes is crucial! There are three operator domains, as shown in the figure below, and these operators control promoters PR and PRM which initiate transcription in opposing directions. Only one of these two promoters is usually active, and it is a bit of a "contest" to see which one wins. If PR wins and the protein cro is made, then production of cI will be repressed. If on the other hand promoter PRM wins and the protein cI is made, then production of cro will be repressed.
Wow! Is that simple or what!
If cro predominates, it hogs the operator region and prevents cI from being made. On the other hand if cI predominates, it hogs the operator region, causing more of itself to be made (from the PRM promoter).
I can hear you asking... how is cI made, since the initial transcription from the genome seems only to be going away from cI? There is a promoter I haven't mentioned called PRE that is activated by cII and cIII (which are produced after the anti-terminator N is made).
The "decision" on how much cI should be synthesized depends on the host of the cell and the number of infecting viral particles. Ultimately, either the cI or the cro will predominate, and one of the following two patterns of gene expression will result:
Obviously the first pattern leads to the growth of the virus (only a fraction of the genes involved in lysis are actually shown) and the death of the cell. The second pattern is the more interesting for our purposes. In the lower panel, cI is the only gene that is being expressed in the virus, and it is involved in a positive feedback loop to induce more of its own expression.
This explains why the lysogenic state is stable. The genome of the virus is essentially shut down during lysogeny, except for a single repressor protein. If another lambda happens to come along it's out of luck! The cI repressor from the first lambda simply prevents expression of the second lambda genome, and it fails to enter a lytic cycle. That explains bacteriophage immunity!
One allele of cI that is important in the laboratory is cI857, which is temperature sensitive (the protein is active at 32 degrees centigrade but inactivated at 39 degrees centigrade). We may therefore grow a lambda phage carrying cI857 as a lysogen at low temperature, then induce lytic growth by simply moving it to a warmer incubator. Very cool!
Lambda can be engineered to carry DNA into cells.
|Do you recall the definition of bacterial transformation? That's when bacteria take up free DNA from their surroundings. If a virus injects its genome into a cell, that's clearly a different type of "uptake" of DNA (or RNA, as the case may be) - we would call that "infection" of course. What term do we use when the infecting virus is carrying DNA that is not normally its own? We call it "transduction" and with integrating viruses like lambda we distinguish between the transfer of genomic DNA adjacent to the normal integration site (specialized transduction) and tranfer of essentially random fragments from the E. coli genome (generalized transduction).|
Let us look at an example of a vector designed to carry foreign DNA - a sort of "engineered transduction". Note the gene marked "434" in the figure, and containing a unique EcoRI site.
The "434" refers to an immunity type of the cI repressor (there are different lambdoid phages in the world, with different classes of immunity, which you may be relieved to know isn't important for this discussion). In any case, the unique EcoRI is a marvelous cloning site in this phage, and an insertion causes disruption of the cI (434) gene. How much foreign DNA can be inserted at the site? The packaging limit is 78% to 105% of the wild-type genome size of lambda, which is 48 kbp. Since lambda gt10 is 43.34 kbp in size, we can fit in an additional 0 to 9 kbp of foreign DNA. If the cI (434) gene is interrupted, then the phenotype will be "clear" plaques, right?
When we plate the recombinant phage, we can see whether each one carries a foreign DNA insert by simply looking to see whether each plaque is turbid or clear. A clear plaque indicates an interrupted cI gene, just as a white colony indicated an interrupted lacZ gene in our previous discussions. In fact, we can go one step further and plate the cells on an hfl mutant strain of E. coli to select against lytic growth of non-recombinant lambda gt10 phage. The "hfl" stands for "high frequency of lysogeny" and when temperate lambda is plated on an hfl strain, there is near certainty that it will grow lysogenically rather than lytically. If the cI gene is interrupted (in this case, by the DNA you have inserted at the EcoRI site) then it will grow lytically even on an hfl strain of E. coli. This is therefore a handy way to select against those phage that lack insertions - they will grow only lysogenically on hfl E. coli, and so will not generate any progeny phage.
Lambda vectors can accept larger pieces of DNA than traditional plasmid vectors, and that is a tremendous advantage. On the other hand, one cannot efficiently introduce lambda DNA into a bacterium by transformation because the 50 kbp size makes it sensitive to shearing or breaking. (It should be noted that infecting a cell by transforming it with DNA, a process called transfection by bacteriologists, is not impossible -- it just isn't feasible on a grand scale)
How then, do we introduce the lambda DNA into cells? We can package the ligated DNA into viral capsids in vitro, then infect the cells with the newly created infectious particles. There are several commercial sources of "packaging extracts" that permit you to package ligated lambda concatamers into viral capsids.
The process is detailed in the figure below:
The packaged lambda phage can then immediately be combined with E. coli, to introduce the recombinant phage vector efficiently into cells.
If that seems pretty complicated, look at this easy protocol that makes use of the Stratagene Gigapack kit (a two-lysate method):
Now that you've seen an example of a lambda cloning vector (gt10), and have learned about the handling methods for packaging of DNA into viral capsids, let us go on to discuss another example of a lambda vector.
Consider the vector lambda gt11, shown below, which has a cloning site at a unique EcoRI (at 19.60 kbp) in a lacZ gene:
The size of gt11 is approximately 43 kbp, so the vector can accept insertions of 0 to 9 kbp.
There's our old friend lacZ again! In this phage vector, interruption of the lacZ gene is the hallmark of successful cloning, and we add IPTG and X-gal to the bacteriological plate to develop the blue-white screening method:
The only difference between this assay and what we discussed in the previous lecture is that the color develops in a plaque (area of lysed bacteria) rather than in a colony.
A slightly fancier version of gt11 is called lambda ZAP II, and it can be obtained from Stratagene.
In either lambda gt11 or ZAP II, DNA sequences containing open reading frames are fused to the lacZ open reading frame.
The result is a chimeric (split) gene, which may encode a fusion protein (provided that the orientation and reading frames of the lacZ and insert are matched):
If the inserted DNA is matched in orientation and reading frame with the lacZ gene, so that it becomes expressed as a fusion protein in E. coli, then we can screen the plaques using antibodies directed against the foreign protein (i.e. encoded by the inserted DNA) as a probe.
In which we find an answer
Here is a summary of how you can use this method practically to answer the BIG question.
Starting with a pure protein, how can you find the gene encoding it?
One important point to consider, when expressing foreign proteins in E. coli, is that many introduced sequences turn out to encode proteins that are toxic to the bacterium. That is, your clone may kill itself if the fusion protein is expressed! Yikes!!! There's a way to solve this little problem, and it has to do with keeping control of the operators.
The trick for screening is to plate the infected cells at 32 degrees centigrade and allow them to propagate as lysogens for a few hours. Then, move the plates to a 39 degree incubator and simultaneously apply a filter soaked in IPTG. The higher temperature destroys the cI857 protein (remember - it's temperature sensitive), leading to lytic growth of the virus, and the IPTG derepresses the lac operon so that lots of fusion protein is expressed and released into the developing plaque.
Department of Biology
California State University Northridge
Northridge CA 91330-8303
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