M13 Bacteriophage Based Cloning Vectors

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 (6^th 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)