Virulent lambda vectors
| Lambda FIX - from Stratagene Inc. |
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http://www.stratagene.com/vectors/maps/lambda/lambda_fix_2.gif |
Note that there appear to be two polylinkers; one at 20.00 kbp and the other at 32.78 kbp. In fact, the sequence between the polylinkers (ninL44, bio, etc.) is a "stuffer" fragment that is discarded. The purpose of the stuffer fragment is just to serve as a "placeholder" while the vector is being replicated as a phage.
Don't forget that lambda phage are only viable if they contain between about 39 and 52 kbp of DNA. With the 14 kbp stuffer present, the FIX sequence would amount to 43 kbp which would make a viable phage. Without the stuffer, the remaining 29 kbpwould be too small to make a viable phage! The stuffer is really like that little piece of white cardboard under the "Twinkee." The cardboard helps the product keep its shape, but when you're ready to eat, you throw it away.
Where did the cI gene go? It was left out! Everything not needed for virus production was eliminated from this vector, so that there would be extra room for foreign DNA inserts. Because of this consideration, the amount of lambda DNA in the two arms is 29 kbp, leaving up to 23 kbp free (because 29 kbp + 23 kbp = 52 kbp, which is the maximum size). What is the consequence of leaving out these sequences? The virus can only grow lytically.
The cloning steps used with lambda FIX are exactly the same as described previously for lambda ZAP (and gt11, and gt10). You prepare fragments of DNA using one of the enzymes shown in the polylinker (or at least arrange to add appropriate linkers or adapters), ligate the fragment(s) into the prepared arms of the phage, and package the resulting concatemers into phage capsids.
Cosmids
You can never have enough room to clone your favorite piece of DNA, it would seem! What prevents us from simply taking over all of the space lambda could offer in its viral capsid? If we could just fill the capsid with a big plasmid having cos ends (necessary for packaging) then we would have about 42 kbp of free space instead of only 23 kbp (as in lambda FIX). In fact, that kind of cloning vector has been made already, and it is called a "cosmid" (where the "cos" indicates that it has lambda cos ends). Here's an example of a commercial vector based on cosmid technology:
| SuperCos I - from Stratagene Inc. |
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Let us look at the anatomy of this vector:
- A sequence marked "ori" for DNA replication in bacteria
- Ampr for ampicillin selection in bacteria
- A sequence marked MCS (multiple cloning site) that is a polylinker containing unique restriction sites and two phage RNA polymerase promoters (T7 and T3) in opposing directions.
- Two cos sequences, separated by an Xba1 site
- An origin of DNA replication from SV40 (permits replication and copy number amplification in many eukaryotic cells, in the presence of SV40 T antigen protein)
- Neor for selection in eukaryotic cells with the neomycin antibiotic analog G418.
The maximum amount of DNA that can be inserted into SuperCos I depends on the packaging limit of lamba (52 kbp) and the pre-existing size of the vector (7.6 kbp).
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How much space does SuperCos I have for foreign DNA? |
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52 kbp - 7.6 kbp = 44.4 kbp |
Bacterial artificial chromosomes
We have had an opportunity to learn about several methods by which DNA is taken up naturally by E. coli:
- Transformation
- Transduction
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.
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male bacterium, showing off his pili |
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.
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Conjugation in bacteria - transfer of an F factor |
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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!
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Conjugation in Hfr bacteria - transfer of a chromosome fragment |
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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.
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:
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pBACe3.6 |
 http://www.chori.org/bacpac/pbace36.htm |
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description of pBACe3.6: The new vector has been named pBACe3.6. Specifically, we maintained the wildtype loxP site, added an additional mutant loxP511 site, a site for the intron encoded nuclease PI-SceI and the Tn7att site. In addition, we changed the cloning area to allow positive selection for inserts containing BAC clones through inclusion of the sacBII gene from Nat Sternberg's Pl vector (Pierce et al. 1992, Proc. Natl. Acad. Sci. USA 89: 2056-2060) and disrupted the BamHI cloning site with a fragment containing a pUC plasmid. The presence of this pUC plasmid serves dual functions: high copy number of the vector for preparing large quantities and appropriate disruption of the sacBII gene to increase viability of the vector containing strain. In addition to the BamHI site, 5 additional sites can be used for preparing BAC libraries: SacI, SacII, MluI, EcoRI and AvaIII. The EcoRI site is particularly important in view of the use of this site in the RARE cleavage procedure, thus opening the possibility for selective cloning of similar fragments from different DNA donors. |
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.
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At left is an example of such a filter (not from RPCI however) from the Cambridge
group's project with Trypanosoma. Note that each dot represents DNA from a different
PAC! (approximately 80 kbp of genomic sequence in each dot) The PACs are stored as bacterial strains, in a freezer, so you can always make more. |
| Here are some comments from RPCI,
a supplier of gridded libraries: All of the RPCI genomic DNA libraries have been gridded onto 22x22cm positively charged nylon filters for hybridization screening purposes. Each filter contains 36,864 colonies which represent 18,432 independent clones that have been spotted in duplicate in a 4x4 clone array. Filter sets are available directly from BACPAC Resources, Department of Human Genetics, Roswell Park Cancer Institute. Filters should be hybridized with (32)P-labeled (random-primed) probes (1x106 cpm/ml hybridization solution) using standard conditions. After washing to remove unbound probe, filters are wrapped in plastic film (Saran) and exposed to x-ray film (Kodak Xar, Amersham Hyperfilm) for 2 to 24 hrs. Complete documentation on interpretation of hybridization results (location of positive clone plate coordinates) is included with the filter sets. Positive individual clones are available for $10/clone shipped using purchasers Federal Express account number. Contact Joe Catanese or Pieter de Jong with requests. |
