The most essential requirement for any cloning
vector is that it has a means of replicating in the host cell. For plasmid
vectors this requirement is easy to satisfy, as relatively short DNA sequences
are able to act as plasmid origins of replication, and most, if not all, of the
enzymes needed for replication are provided by the host cell. Elaborate
manipulations, such as those that resulted in pBR322 , are therefore possible
so long as the final construction has an intact, functional replication origin.
With bacteriophages such as M13 and λ, the situation
as regards replication is more complex. Phage DNA molecules generally carry
several genes that are essential for replication, including genes coding for
components of the phage protein coat and phage specific DNA replicative
enzymes. Alteration or deletion of any of these genes will impair or destroy
the replicative ability of the resulting molecule. There is therefore much less
freedom to modify phage DNA molecules, and generally phage cloning vectors are
only slightly different from the parent molecule.
Bacteriophages replicate by two processes or cycles- lytic and
lysogenic.
Construction of a phage vector:
The normal M13 genome is 6.4 kb in length, but most
of this is taken up by ten closely packed genes, each essential for the
replication of the phage. There is only a single 507-nucleotide intergenic
sequence into which new DNA could be inserted without disrupting one of these
genes, and this region includes the replication origin which must itself remain
intact. Clearly there is only limited scope for modifying the M13 genome.
Figure : The M13 genome showing
genes I to X.
The first step in construction of an M13 cloning
vector was to introduce the lacZ′ gene into the intergenic sequence.
This gave rise to M13mp1, which forms blue plaques on X-gal agar (Figure 6.6a).
M13mp1 does not possess any unique restriction sites in the lacZ′ gene.
It does, however, contain the hexanucleotide GGATTC near the start of the gene.
A single nucleotide change would make this GAATTC, which is an EcoRI
site. This alteration was carried out using in vitro mutagenesis ,
resulting in M13mp2.
Figure : Construction of the M13mp1 and M13mp2 vectors from the
wild type M13 genome.
M13 mp1
Characteristics of the M13mp1:
·
Forms blue plaque on Xgal
plate.
·
No unique restriction site
on lacZ’.
·
Contains the hexanucleotide
GGATTC near start of gene.
·
A single nucleotide change
would make it GAATTC, the recognition sequence for EcoR1.
·
This alteration was carried
out by in-vitro mutagenesis and gave rise to M13mp2 vector.
M13mp2
Characteristics of the M13mp2:
·
Simplest M13 cloning vector.
·
It has a slightly altered
lacZ’ gene (6th codon codes for Asparagine instead of Aspartic Acid)
but β galactosidase enzyme produced by cells infected with M13mp2 is still
perfectly functional.
·
DNA fragments with EcoR1
sticky ends can be inserted into the sticky ends and recombinants are
distinguished as clear plaques on Xgal Agar.
M13mp7
Figure :
Construction of M13mp7
After M13mp2 formation, additional restriction sites were introduced
in lacZ’ gene by a polylinker to give M13mp7. A polylinker is a short
oligonucleotide sequence with unique restriction sites and EcoR1 sticky ends. It
has 4 possible cloning sites EcoR1, BamH1, Sal1, and Pst1. The polylinker is
designed in such a way that it doesn’t totally disrupt the LacZ’ gene. It is
functional though altered; β -galactosidase enzyme is still produced. When
M13mp7 is digested with EcoR1, BamH1, Sal1or Pst1 a part or entire polylinker
is excised. On ligation 3 things may occur:
-
New DNA is inserted
-
Polylinker is reinserted
-
Vector self ligates without
insertion.
Insertion of a new DNA almost invariably prevents β-galactosidase
production so recombinant plaques are clear on Xgal Agar. If polylinker is reinserted
and original M13mp7 reformed then blue plaques result. Self-ligation always
results in a functional lacZ’ gene giving blue plaques. Another important
feature of M13mp7 is that DNA inserted into EcoR1, BamH1, Sal1or Pst1 can be
excised using EcoR1. Very few vectors allow cloned DNA to be recovered so
easily.
pEMBL8
Figure : pEMBL8 , a hybrid
M13 vector
Although M13 vectors are very useful for
the production of single-stranded versions of cloned genes, they do suffer from
one disadvantage. There is a limit to the size of DNA fragment that can be
cloned with an M13 vector, with 1500 bp generally being looked on as the
maximum capacity, though fragments up to 3 kb have occasionally been cloned. To
get around this problem a number of hybrid vectors (“phagemids”) have been
developed by combining a part of the M13 genome with plasmid DNA.
An example is provided by pEMBL8, which
was made by transferring into pUC8 a 1300 bp fragment of the M13 genome. This
piece of M13 DNA contains the signal sequence recognized by the enzymes that
convert the normal double-stranded M13 molecule into single-stranded DNA before
secretion of new phage particles. This signal sequence is still functional even
though detached from the rest of the M13 genome, so pEMBL8 molecules are also
converted into single-stranded DNA and secreted as defective phage particles. All that
is necessary is that the E. coli cells used as hosts for a pEMBL8
cloning experiment are subsequently infected with normal M13 to act as a helper phage, providing
the necessary replicative enzymes and phage coat proteins. pEMBL8, being
derived from pUC8, has the polylinker cloning sites within the lacZ′ gene,
so recombinant plaques can be identified in the standard way on agar containing
X-gal. With pEMBL8, single-stranded versions of cloned DNA fragments up to 10
kb in length can be obtained, greatly extending the range of the M13 cloning
system.
( Notes from: TA Brown 6th Edition)