Virulent lambda vectors
Size does matter
How many different recombinants must a library have?
Let us carry on this discussion about the representation of libraries. In the last lecture I mentioned that since a library contains random fragments, you can never be 100% certain that it contains a clone you desire.
No, with only "one covering" on hand, they only keep 1-(1/e) dry. If you are an ant on the big golden carpet, there is a 63% chance that you are being kept dry. If the number of umbrellas is doubled, the protection from the rain goes from 63% to 86%, as 1-(1/e)2
Sounds pretty fishy to me, but that's the Poisson distribution.
With our analogy of clones, the uncovered regions represent uncloned sequences in the genome. The way we can calculate what's missing is pretty easy -- it's based on the Poisson distribution. If we have one "covering" of the genome (as in the ten clone example) the chance of a sequence being cloned is (1- 1/exp(1)) or about 0.63 (or 63%), where exp(x) = the base of the natural logarithm e to the x power, . If we have two "coverings" of the genome (as in the twenty clone example) the chance of a sequence being cloned is (1- 1/exp(2)) or about 0.86 (or 86%), where exp(2) means the square of e. The pattern continues as your library contains more independent clones. With ten "coverings", the chances reach 0.99995 (or 99.995%), but you can see that you never reach absolute certainty of cloning any particular sequence.
|If I have a library with 100 phage in it, and I grow it in bacteria so that, following lysis, I have 10,000 phage, will I have a better chance of getting a clone I want?||
No, your chances are not improved. The issue of representation in a library deals with a count of the independent phage clones. If you make copies of the original set, you still only have 100 independent phage, even if you "amplify" the library so your tube now has 10,000 phage.
Here are a couple of problems to think about:
Virulent lambda vectors
|We've already seen several advantages to having a temperate phage
vector. In the case of lambda gt10, the ligation products lacking insertions can
be selected or screened out by virtue of their lysogeny. In the case of lambda gt11
and ZAP vectors, maintaining a clone in a lysogenic state can minimize expression
of a potentially toxic protein product from the inserted sequence. Sometimes, cloning
pieces of DNA 9 kbp at a time is just too impractical. To clone bigger pieces (9
to 23 kbp) you need a "stripped down" version of the phage. For example,
a look at lambda FIX.
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 kbp would 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, using steps 1-6 in the figure below.
Reminder: The split life cycle of wild type lambda
Since lambda FIX cannot use steps 7-9 of the life cycle, it is now purely virulent, and no longer temperate.
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.
Two legitimate concerns:
What happens if the two phage arms ligate together
without an insert between them? Nothing! The two arms combined
are too small to be packaged into a viable phage, so you automatically select for
phage with insertions.
What happens if the stuffer is recloned in the
phage arms, instead of your added DNA fragment? This is certainly
a problem, but there are several solutions.
What trick might rescue us from the drudgery of screening clones with reinserted stuffers? Another sleight of hand with bacteriophage. It turns out that E. coli strains lysogenic for P2 bacteriophage (another temperate phage we haven't talked about) are unwilling to countenance having bacteriophage lambda in the same cell. It seems that P2 interferes with lambda infections because of the lambda red/gam genes. Lambda that are red/gam+ are Sensitive to P2 Interference (said to have the Spi+ phenotype). Guess what? The red/gam genes are present in the stuffer fragment of lambda FIX! That means that lambda FIX carrying a re-inserted stuffer (i.e. those we don't want in our collection of clones) will be Spi+, and will therefore not grow on a P2 lysogenic strain of E. coli. Those lambda FIX carrying foreign DNA instead of stuffer will be Spi-, and will grow on a P2 lysogenic strain of E. coli. Very cool!
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:
Let us look at the anatomy of this vector:
Numbers 1-3 are what we discussed at the beginning of the course, as being necessary elements. Number 4 permits packaging of the plasmid into lambda phage capsids in an in vitro packaging system. Numbers 5 and 6 are analogous to 1 and 2, but work in eukaryotic cells (some of the time at least). This is a new concept for us - a plasmid that works in two different types of cells. We call this a shuttle vector, because it can shuttle back and forth between the hosts. Of course, we don't use bacterial transformation or phage transduction methods to introduce DNA into eukaryotic cells. We'll discuss the differences later in the course.
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).
If we want to package more than about 45 kbp, we'll have to turn to a phage other than lambda! Bacteriophage P1 is now commonly used as a cloning vector, and has several interesting features. The phage lysogenizes bacteria (it is temperate) but it doesn't usually integrate its DNA into the E. coli genome. Instead, it maintains itself as a plasmid. When it enters a lytic cycle, it makes many copies of its genome which have to be disentangled after replication:
This results in tangled plasmids that need to be resolved into individual unlinked monomers. The protein cre, which is a gene product of phage P1, finds "lox" sites in each of the tangled genomes and causes recombination. After two rounds, the plasmids are untangled.
With that knowledge, let's study the structure of a P1-based vector from RPCI in Buffalo. These vectors can take large insertions of approximately 80 kbp.
Here's an example of a vector for P1 packaging:
It is important to point out some of the features of this vector:
Here is a flow-chart showing how this can be used:
A P1 packaging system can be used for transduction of P1 phage-based plasmids, however there are also P1 artificial chromosomes (called PACs) that are larger than the packaging limit and are simply based on the replicon of P1. These may be handled by transformation.
I'll attempt to decode their technical notations:
Department of Biology
California State University Northridge
Northridge CA 91330-8303
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