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Bacterial Artificial Chromosome (BAC) and fosmid vectors were developed
by Shizuya1 and Kim, UJ2 to
solve the problems of chimerism in Yeast Artificial chromosomes (YACs)
and genomic DNA instability in multicopy cosmid vectors. They showed
that DNA that underwent rearrangements and deletions in cosmids was stable
in BACs and fosmids, which replicate using the single-copy F-factor replicon.
Thus, these vectors have become standard tools for preparing genomic
libraries. EPICENTRE offers both types of single-copy vectors. The pIndigoBAC-5
Cloning-Ready Vectors are BamH I or Hind III restriction
enzyme-cut and completely dephosphorylated, ready for preparation of
high quality BAC libraries. The EpiFOS™ Fosmid Library Production
Kit contains a ready-to-use fosmid vector and all other reagents required
for researchers to prepare up to 10 primary fosmid libraries. The kit
uses a novel blunt-end cloning strategy that results in complete and
unbiased libraries.
The development of vectors with both single-copy and high-copy origins
of replication.
The development of single-copy BAC and fosmid cloning vectors was an
important advance for genomic cloning. However, it is harder to get sufficient
yields and purity of clone DNA from cultures containing single-copy vectors
for sequencing or other applications. This problem was solved by the
laboratory of Dr. Waclaw Szybalski.3-5 These
researchers developed vectors that contained both the single-copy E.
coli F-factor replicon and an inducible high-copy oriV origin
of replication. Since initiation of replication from oriV requires
the trfA gene product, which is not encoded by the vector or by E.
coli strains commonly used for cloning, they also constructed a new E.
coli host strain by inserting the trfA gene joined to a tightly
regulated inducible promoter into the chromosome of the host cell. In
the absence of the inducer of the trfA gene, the new vectors replicate
at one copy per cell using the F-factor replicon. In the presence of
the inducer, the trfA gene is induced and the vectors replicate
using the high-copy oriV. EPICENTRE has incorporated and expanded
upon the Szybalski laboratory’s patented technology in its CopyControl™ BAC
Cloning Kits and its CopyControl™ Fosmid Library Production Kit.
CopyControl™ Cloning Systems provide the benefits of both single-copy
and high-copy vectors.
Genomic libraries constructed using CopyControl BAC and Fosmid Systems
have all of the advantages of both single-copy and high-copy cloning
vectors, without the disadvantages of either. CopyControl clones are
grown at single-copy-number, to ensure insert stability and successful
cloning of sequences encoding toxic proteins, and then, whenever desired,
the clones can be induced to high-copy-number in <2 hours, for high
yields and higher-purity DNA. Extensive analysis by EPICENTRE scientists
of numerous randomly-picked BAC (up to 200-kb) and fosmid clones has
shown that the insert DNA is stably maintained during at least one hundred
generations at single-copy and during the subsequent short (<2-hour)
induction step. CopyControl BAC and Fosmid clones are inducible to 10-20
copies per cell or up to 50 copies per cell, respectively. Greater amounts
of induced BAC or fosmid DNA facilitates purification using smaller cultures
and simpler methods.
How the CopyControl cloning and clone induction process works
The process for using CopyControl BAC or fosmid systems is summarized
in Figure 1 (also see here).
Genomic DNA is first ligated into a CopyControl pCC1BAC™ or a
pCC1FOS™ Vector that contains both the single-copy E. coli F-factor
replicon and an inducible high-copy oriV origin of replication.
If the DNA is ligated into the pCC1BAC Vector, it is then transformed
into TransforMax™ EPI300™ E. coli cells (with or
without phage T1-resistance), improved strains constructed at EPICENTRE
that contain a chromosomal copy-up mutant trfA gene under the
tight control of an inducible promoter. Then, transformants are plated
on LB-chloramphenicol plates for selection. If the DNA is ligated into
the pCC1FOS Vector, it is packaged using high-efficiency lambda packaging
extracts and plated on selection plates with TransforMax EPI300-T1R cells.
In both cases, the resultant transformant clones in the library replicate
in the cells at single copy because expression of the trfA gene
is repressed. CopyControl BAC or fosmid libraries can be maintained
indefinitely at single copy for clone stability. CopyControl BAC or
fosmid clones from the library can be picked and grown individually
in small-volume cultures, and induced to high-copy-number at any time.
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Figure 1. CopyControl™ Cloning Process
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Existing BAC and fosmid clones in standard single-copy vectors, such
as pEpiFOS™-5 or pIndigoBAC-5, can be quickly and easily “retrofitted” to
be inducible to high-copy-number by “hopping” an inducible
high-copy oriV into the clone using a simple in vitro transposition
reaction (see EZ-Tn5™ <oriV/KAN-2> Insertion
Kits).
References
- Shizuya, H and Kouros-Mehr H et al. (2001) Keio J Med 50,
26.
- Kim, UJ et al. (1992) Nucleic Acids Res 20,
1083.
- Hradecna, Z et al. (1998) Microbial and Comp. Genomics 3,
58.
- Wild, J et al. (2001) Plasmid 45, 142.
- Wild, J et al. (2002) Genome Research 12, 1434.
* Covered by issued and pending patents. By
purchasing CopyControl systems or vectors, the purchaser receives the
right to use the product purchased from EPICENTRE or an authorized distributor
only for life science research.
Figure 2 shows schematics of the processes for
constructing genomic libraries using the CopyControl™ Fosmid Library
Construction Kit and the CopyControl™ BAC Cloning Kits available
from EPICENTRE. Both systems have pros and cons.
Briefly, the CopyControl Fosmid Kit uses a unique strategy for cloning
~40-kb blunt-ended genomic DNA that is obtained by simple and mild shearing.
This method does not depend on the presence of restriction enzyme sites
and results in a more random and more complete representation of all
of the genomic DNA. The CopyControl BAC Cloning Kits currently available
all require optimizing and performing partial restriction endonuclease
digestions of the genomic DNA to be cloned.
Fosmid-size DNA inserts are easier to handle and clone than the larger
BAC inserts. The genomic DNA that is cloned using the CopyControl Fosmid
Kit can be purified on an agarose electrophoresis gel, whereas the genomic
DNA for BAC cloning is usually purified by pulsed-field gel electrophoresis.
Following ligation of the genomic DNA into the vector, BAC clones are
obtained by electroporation of electrocompetent E. coli cells,
whereas fosmid clones are obtained by packaging using bacteriophage lambda
packaging extracts and transfecting the E. coli cells. Since the
latter method results in a ~10-fold higher cloning efficiency than transformation
of BAC clones, this step for constructing fosmid libraries is easier
and faster than the transformation step for BAC libraries.
Figure 2.
Panel A. CopyControl™ Fosmid Library Production Kit |
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Panel B. CopyControl™ BAC Cloning Kits
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To meet the genome-coverage requirements of your project, you will need
more clones and more space to store individual clones for a fosmid library
than you will need for the larger BAC clones. If the library will be
used for a sequencing project, you will also need to analyze and assemble
the sequence from more fosmid clones than you would for BAC clones.
Another consideration is that the ~40-kb CopyControl Fosmid clones can
be induced up to ~50 copies per cell, whereas ≥100-kb BAC clones are
usually induced to ~10-20 copies per cell.
The processes for making genomic libraries using the CopyControl Fosmid
Library Construction Kit or a CopyControl BAC Cloning Kit are compared
in Table 1. As a general rule, fosmid cloning will save time and labor
in constructing the library, while BAC cloning may save time and labor
in analyzing the clones. If the genome size is small, the CopyControl
Fosmid Library Construction Kit will probably be the best option. For
cloning larger genomes, the choice may depend on the downstream applications
of the library.
Table 1. A comparison of the CopyControl™ Fosmid
and the CopyControl™ BAC cloning processes.
| CopyControl™ pCC1FOS™ provided linearized at Eco72
I (blunt) site, dephosphorylated, and purified. |
CopyControl™ pCC1BAC™ vector
provided linearized at either the BamH I-, EcoR I-,
or Hind III-site, dephosphorylated, and highly purified. |
| Random shearing followed by end-repair to 5’-phosphorylated,
blunt-ended DNA. End-repaired DNA is size-selected by standard agarose
gel electrophoresis. |
Partial restriction endonuclease digestion. DNA fragments are size-selected
by pulse field gel electrophoresis (PFGE). |
High efficiency lambda phage
packaging followed by infection of TransforMax™ EPI300™ cells. |
Electroporation of high efficiency TransforMax™ EPI300™
Electrocompetent E. coli. |
| Virtually 100% white colonies. |
Greater than 95% white colonies. |
| All inserts are approximately 40 kb. |
Typically an average size of 80 kb to
200 kb. |
| Approximately 10-fold greater than BAC cloning. |
Approximately 10-fold lower than fosmid cloning. |
| Inducible from single-copy to 10-50 copies per cell. |
Inducible from single-copy to 10-20 copies per cell. |
Please contact EPICENTRE’s Technical Consultants to discuss your
projects. We would appreciate to learn your ideas, thoughts, and experiences
related to the two systems.
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