Application of M13 DNA as a Cloning Vector in Protein Engineering

Welcome to my posts about protein engineering. In previous post, we studied about different type of mutations and their effects on amino acids sequence of protein. In this post let’s study about Application of M13 DNA as a Cloning Vector in Protein Engineering.

Tools Use in Protein Engineering

In protein engineering we can apply different methods of Directed Mutagenesis in order to acquire desired characteristics in protein of interest. We need cloning vector to insert gene of interest before subjecting to introduce desired mutation. These cloning vectors can be M13 ssDNA and/or Plasmid DNA etc. Methodologies used in Protein Engineering are;

1. PCR based such as (Amplification, Error-Prone PCR, Degenerate Oligonucleotide Primers).
2. some are nonspecific methods such as (Random Insertion/Deletion Mutagenesis, DNA Shuffling).
3. In parallel, incorporation of unusual amino acids in native proteins are commonly practiced methodologies as well as frequently apply in Protein engineering.

What is Cloning Vector?

Cloning vector is a small molecule of DNA, that can be stably maintained in host organism along with host genomic DNA. In addition, a foreign DNA, not belonging to the organism from which the vector has been isolated, fragment can be inserted for cloning purpose, this type of molecule is known as cloning vectors. Number of Plasmids of bacteria and yeast as well as viral genomes are used as cloning vectors. M13 genome has also been developed to use as a cloning vector by inserting the following elements.

1.  lac repressor gene (lac I),
2. Operator region of lacZ gene
3. And a polylinker site that is used to insert gene of interest for cloning purpose.

What is M13 DNA?

Before going to explain M13 DNA mediated oligonucleotide directed mutagenesis. We should know about what is M13 DNA?

Morphology of the M13 Phage

Being a Protein Engineering reader, we should know at least the following characteristics of M13 virus. M13 has;

Shape and Type of M13 Virus

Morphologically, M13 is a filamentous bacteriophage. Bacteriophages are bacteria infecting viruses. Being bacteriophage, M13 infects E. coli bacteria.

Size of M13 Virus

Size of M13 phage is dependent of the size of genomic DNA. Genomic DNA dependent size of M13 was confirmed by an experiment, in which approximately 42 Kilo bases DNA fragment (approx. 7X of M13 genome ) was inserted into M13 genome. It was easily packaged in filament of M13 virus.

M13 Genome

1. Circular  ssDNA (ssDNA means singal stranded DNA)
2. 6.4 kilo nucleotides long
3. The M13 genome consists of total 10 genes. These genes are denoted by Roman numerical digits. I, II, III,………….upto X

M13 Protein Coat

Like other protein coated viruses, M13 genome is also encapsulated by protein coat. Protein coat is made of two different types of proteins, product of two different genes (VIII and VIII).

1. Gene VIII coded protein forms cylindrical array of approximately 2,700 identical subunits surrounding the viral genome.
2. The product of gene III, approximately 5-8 copies, are present at the end of the filamentous phage. Minor protein coat allows binding to bacterial “sex” pilus

How M13 Can Deliver our Gene of Interest to Host cell?

To understand how M13 deliver gene of interest to host cell (E. coli)? we should know about mechanism of infection of E. coli by M13 virus. As I mentioned above that minor coat protein helps M13 virus to attach to sex pilus of E.coli during infection. After binding to sex pilus, M13 virus injects its genomic DNA into host bacteria, E. coli.

Major coat proteins are stripped off on the surface of E. coli while minor coat protein remain attached. As the genomic circular ssDNA molecule enters the cytoplasmic region of host cells. Host replicative machinery convert ss (+) DNA into double stranded circular DNA. This molecule is known as “Replicative Form” or Simply RF.

Amplification of M13 Genome

How M13 genome is amplified in host cells? After inserting circular ssDNA into host bacteria. Replicative machinery of host cell converts it into dsDNA molecule known as Replicative form or (RF). Replicative form consists of two strands, denoted by “+” and “-“. The product of Gene II works like restriction enzyme and makes cut that is known as ‘nick’ in “+” strand of RF.
At this stage, an enzyme known as “DNA Polymerase 1” extends the other strand. As DNA polymerase 1 mediated amplification is completed around the whole genome. Then product of gene II cuts again the same strand to release a completed copy of “+” genome in linear form. Later this “+” strand is circularised. “+” strand genome is converted into double stranded (RF) within 15-20 minutes as replication of DNA started.

How Amplification of M13 is Inhibited?

In the meantime, Gene V synthesised pV protein. This is ssDNA binding protein and responsible for the prevention of RF formation by single (+) strand. In this way, further replication of M13 genome stopped at this stage.

Reconstruction of M13 Virus

Products of genes II, III and V are responsible for M13 genome packaging.

1. After nicking by gene II protein that is responsible to covert replicative form into ssDNA, gene V proteins attaches with ssDNA molecule and carries DNA molecule toward plasma membrane of host cell. 
2. At this stage, major coat protein of M13 is present in E. coli membrane. Here, Product of gene V is shaded off and the major coat proteins encapsulate phage DNA as it is leaving the host cell.
3. Gene III product (approx. eight copies attached at the end) helps in extrusion of M13 genome from host cell (E. coli) surface.
4. Now the viral particle is ready to infect another host cell.

These properties make M13 as a suitable molecule to use as a cloning vector. We can use M13 ssDNA as a cloning vector to clone the inserted gene with specific mutation.

How is it possible to mutate the inserted gene in M13 ss DNA?

Inserted gene in ssDNA of M13 virus can be mutated by using the following steps.

1. First insertion of gene of interest which we want to mutate using ssDNA of M13 as a template.
2. Then replication of the other strand to convert it into dsDNA using mutated oligonucleotide primers and Klenow fragment as Polymerase enzyme. Note: Klenow fragement avoids repairing mutation in the amplified DNA molecule.
3. Then ligation of second strand with T4 DNA ligase and transformation to specific host. T4DNA ligase is used to fill the gap by establishing phosphate bond between 5′-P and OH-3′ group. The main objective of this step to avoid degradation of nicked DNA by exonuclease activity of host enzymes.
4. Now in host we have two type of cloning vector;
   1. One vector with normal version of gene of interest
   2. While other one with desired mutation in gene of interest using mutated oligonucleotide primer.

Products of Gene of Interest inserted in ssDNA of M13 Virus

Using this strategy we can mutate, one copy of gene of interest. So, during amplification we will face both normal gene product and desired mutated gene product. Both will be in mixture with approximately 50% ratio.

How to enrich the cloning vector with desired mutation? we will study in next post.