ligation/transformation (type I restriction)
HARDIES at THORIN.UTHSCSA.EDU
HARDIES at THORIN.UTHSCSA.EDU
Wed Mar 8 00:30:47 EST 1995
After HARDIES at THORIN.UTHSCSA.EDU wrote:
: Make sure the host strains haven't been switched around on you somehow.
: Your result sounds exactly like you're trying to break through the
: K, B, or P restriction system.
Shin Enomoto asks:
> How often does restriction system interfere if the source of the cloning
involve 2 fragments derived from E. coli? I would imagine this can only
happen if the ligation junction creates a de novo restrcition sites.
This restriction system interference might be more of a problem if one is
working from a PCR product. Is this encountered often?
K, B, or P sites appear about every 1000 bp in essentially every DNA.
Unless the DNA was specifically produced in a modification competent
strain of E. coli, the sites will be unmethylated and hence
vulnerable. If you go into a restriction positive strain of E. coli,
you lose 1000x efficiency for each such site. Unlike the more
familiar Type II restriction sytems, these enzymes recognize the
unmodified site, translocate around the molecule, and cleave it to
shreds. None of the familiar cloning strains protect B or P sites;
some protect K sites and some don't. So there's a good chance you're
working with DNA that is vulnerable to attack by these systems. PCR
products are definitely unmodified; but since they are small, some
will carry a site and some won't.
Consequently, cloning strains have to be engineered to lack any such
system, or else you can't do much of anything. The original E. coli
K-12 had a K system, which has been mutated away in the common cloning
strains. (hsdR-, sometimes called rK-)
So how do you get into trouble?
1. Use JM101, JM102, JM103. These strains were engineered for use
with M13 vectors and the K system was supposed to be mutated away.
But they screwed it up and left it intact. This is how M13 vectors
got the reputation for not being able to carry inserts greater
than a few hundred basepairs.
2. Use some strain not designed for cloning. Most of the classic
E. coli mutant strains carry an active K or B system. People
often decide to clone into one of these to make use of complementation
to the peculiar mutation contained in it. I've seen two different
groups blow over 6 months on this mistake before they figured it out.
3. Start with a cloning strain, try to alter its genetic background
by P1 transduction, screw up and leave a P1 prophage in the modified
strain. The phage carries the P restriction system, so you've just
made your strain incompatible with cloning anything.
4. Maintain you cloning strain by serial propagation year in and year
out. Eventually you'll pick some contaminant that blew into your petri
dish, and it'll have some restriction system, as well as all kinds of
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu
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