Methods and reagents: Reducing background colonies with positive selection
vectors

Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet.  This month's column discusses the pros and cons of eliminating
unwanted background colonies by using the positive selection vector pZErO.
For details on how to partake in the newsgroup, see the accompanying box.

Experienced gene unspoolers will most certainly tell you that the main problem
with cloning genes is the laborious, repetitive and time-consuming screening
process involved.  To locate the one clone containing the correct insert, many
colonies must be screened either by restriction analysis of plasmid mini-prep
DNA, or by amplicon-sizing after the polymerase chain reaction (PCR).

As well as causing a bad case of `eppi-thumb' (a sore thumb caused caused by
repeatedly opening microfuge caps), mini-prep analyses typically reveal colonies
with empty vectors, causing enormous frustration when repeated attempts to get
the desired recombinant DNA molecule are made.

Even when a blue/white colony indicator is used for determining when the
beta-galactosidase gene on the vector is interrupted, readthrough of the cloned
DNA fragment sometimes allows for some expression, leading to leaky phenotypes
and variation in the intensity of blue colonies.  In addition, the blue color of
colonies can vary on indicator plates, depending on the particular supplier and
batch of X-Gal.  This can make the search for the correct colony less
predictable.

Zero Background
***************
Over the past few years, netters have been discussing a new vector system that
eliminates much of the empty vector problem.  By forcing a powerful positive
selection for vectors with inserted DNA, the Zero Background [TM] Cloning Kit
from Invitrogen can reduce the background of empty vectors in cloning
experiments.

The basis of the selection lies in the ability of the ccdB (coupled cell
division) gene from the F plasmid to selectively kill cells that do not have a
corresponding override ccdA gene [1]. The ccdB gene encodes a lethal protein
that poisons topoisomerase II (DNA gyrase) causing unrecoverable DNA damage
within the cell [2], and the ccdA gene encodes the `antidote' to the product of
ccdB [1,3].

Cloning vectors carrying the cloned ccdB gene are cleverly designed to have the
lethal gene under control of the inducible lac promoter.  Insertions of foreign
DNA that disrupt expression of the ccdB gene allow only those plasmids with
inserts to survive transformation.  Selection for the antibiotic resistance gene
on the vector ensures recovery of only recombinant plasmids [4].

Dephosphorylating the plasmid DNA is not necessary as religated vector is lethal
to the cell. This eliminates all the common problems associated with the use of
calf intestinal alkaline phosphatase (CIAP), such as incomplete heat
inactivation of the CIAP and loss of DNA during subsequent organic extractions
and precipitations.

In general, netters say that this system saves a lot of time and frustration
when cloning, because there really is no need to optimize vector-to-insert
ratios. Much of the guesswork involved with determining the amount of DNA to be
used in ligation reactions is unnecessary. They say that one typically gets the
DNA inserted in more than 90% of colonies screened without overworking the DNA,
and that this greatly reduces the screening process.

One hint, suggested by netters, to reduce background even further is to allow
restriction digests to go for a very short time (20-30 min). Flush ends digested
by the enzyme might religate and cause frame shifts within the ccdB gene; these
plasmids would not kill the cell.  Typically, you can expect to see the fragment
of interest by screening less than a dozen colonies.

Sub-Zero
********
Although the kits containing the vectors pZErO1, pZErO1.1 and pZErO2.1 have been
on the market for a few years, netters are now gaining more experience with them
and can offer hints for improving their usefulness in the lab. Netters complain
that the main drawbacks of using the zero background cloning system are the cost
of the kits and media components. For example, the IPTG needed to express the
ccdB gene and the Zeocin needed for plasmid selection are expensive. Media and
solutions containing these compounds also have a limited shelf life.  The
specialized antibiotic Zeocin (a bleomycin-like intercalating compound promoted
by Invitrogen) is much more expensive than most other common antibiotics.
Netters say that even though others have suggested using only half the
concentration of Zeocin in order to cut the cost, they would prefer to use
carbenicillin or a combination of ampicillin and methicillin for reducing
satellite colonies instead. Therefore, they would have preferred the vectors to
be developed with the more common ampicillin-resistance gene.

Zeocin provides another disadvantage in that a low-salt medium is required for
activity, and making the special media is inconvenient.  Zeocin is also
light-sensitive and plates poured in advance do not last more than a week when
stored at 4 degrees C.  Because kanamycin is much cheaper, netters now prefer
the newer pZErO-2.1 vector to the older pZErO2-1.1 vector because it has a
kanamycin-resistance marker instead of the Zeocin marker.  In addition, one
person mentioned that none of the vectors have opposing T7 and SP6 promoters,
and that this makes them less desirable than other common vectors.

Grow your own vector?
*********************
Some people have been wondering how the vector can be propagated in order to
make their own vector DNA if it kills the cell upon transformation. A printed
warning appears with the kit that common laboratory strains of bacteria should
not be used for propagating the vector, but no further explanation is given.
Presumably, upon transformation, cells that escape the poison can either have
mutations within the ccdB gene on the vector, or have permissive mutations
within the gene encoding topoisomerase, such as the gyrA462 mutant already
isolated [2].

However, some netters are willing to live dangerously and have tried their hand
at creating some homemade vector DNA.  Those who have done it say that they had
no problem propagating the vector in the supplied TOP10F' host cells and that
the isolated DNA behaved normally.

With all technical points aside, bulking up the plasmid DNA, if even for your
own use, seems to be covered by a license agreement that must be signed and
returned to the company before you use the kit.  Netters see this as just one
more drawback in that scientists have to buy the vector DNA every time it is
required for an experiment.

One netter, Ferdinand Sven Vilim (vilimf01@mcrcr6.med.nyu.edu) wrote that with
all the imposed restrictions on its use, these vectors would not be his first
choice, especially because he feels a researcher should not have to buy a
particular plasmid vector more than once.  Some feel it is even more unfair that
the published lineages of vectors leading up those developed for this system
[5,6] are also seemingly blocked from public use.  However unsettling that might
be for free-thinking scientists, it seems to be the wave of the future for
scientific research products.

Netters feel that all the legal ownership rights excluding researchers from
propagating or creating their own plasmid DNA, and the requirement to inform the
owner every time you send a recombinant plasmid to a third party, is just too
much and simply unjustified. Many are now refusing to buy restricted-use kits
just because of the extra paperwork involved.

References
**********

[1] Ogura, T. and Hiraga, S. (1983)
    Proc. Natl. Acad. Sci. U.S.A. 80, 4784-4788

[2] Bernard, P. and Couturier, M. (1992)
    J. Mol. Biol. 226, 735-745

[3] Miki, T., Yoshioka, K. and Horiuchi, T. (1984)
    J. Mol. Biol. 174, 605-625

[4] Bernard, P. (1996)
    BioTechniques 21, 320-323

[5] Bernard, P. et al. (1994)
    Gene 148, 71-74

[6] Bernard, P. (1995)
    Gene 162, 159-160

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Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are those of
the author and do not reflect the opinion of the National Cancer Institute or
the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the author.

You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/mar97.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1997. Methods and reagents: Reducing background colonies with
positive selection vectors. Trends in Biochemical Sciences 22(3):138-139.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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