Cloning

Cloning Vector: The Molecular Vehicle

By Somanath Sahoo

Contents

1. ​Introduction to cloning vectors

2. ​Classification, characteristics & significance of cloning vectors

3. Plasmids

4. Bacteriophages (Biology of bacteriophage lambda, Insertion and Replacement vectors)

5. Phagemids

6.Cosmids

7.Artificial chromosome vectors (YACs; BACs)

8.Expression vectors​

Clone: from the Greek - klon, a twig

The production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques.

The DNA containing the target gene(s) is split into fragments using restriction enzymes. These fragments are then inserted into cloning vectors which transfer the recombinant DNA to suitable host cells.

Inside the host cell the recombinant DNA undergoes replication; thus, a bacterial host will give rise to a colony of cells containing the cloned target gene.​ Here the target gene can also express like the host genes.

Introduction

Classification

​1. Plasmid

2. Bacteriophage

3. Cosmid

4. Bacterial artificial chromosome

5. Yeast artificial chromosome

6. Human artificial chromosome

7.​ Phasmids

Self replication, multiple copies

Replication origin site

Multiple cloning sites

Selectable marker gene

Small size: less than 10kb in size

Low molecular weight

Easily isolated & purified

Easily inserted into host cell

Control elements – promoter, operator, ribosome binding site

Characteristics of cloning vectors

A particular gene can be isolated and its nucleotide sequence determined

Control sequences of DNA can be identified & analyzed

Protein/enzyme/RNA function can be investigated

Mutations can be identified, e.g. gene defects related to specific diseases

Organisms can be ‘engineered’ for specific purposes

Significance of cloning vectors

Plasmids

In 1973, Cohen described first successful contruction of recombinant vector. Plasmid PSC101 of E. coli.

Bacteriophages

More potential vector than plasmid vectors

​1. Cloning large DNA fragments

2. Linear phage molecule

3. Efficient than plasmid

4. Used in storage of recombinant DNA

5. Used bacteria (E. coli) as host cell

​

​Use of bacteriophages as a cloning vectors

Biology of lambda bacteriophage

Lambda phage as cloning vector

Escherichia coli as a host cell to multipy the rDNA

​Figure 1. Bacteriophage lambda development.

(1) Attachment of the virus to the cell and injection of the dsDNA genome. The location of the first (A) and last (R) genes in the monomeric linear genome are indicated.

(2) Circularization of the genome forming an intact cos site (dark oval).

(3) Bidirectional, or u replication yields daughter circles.

(4) Rolling-circle, or s replication, gives rise to linear concatemers of the viral genome.

(5) Assembly of a multiprotein pre-nicking complex.

(6) Terminase-mediated duplex nicking and strand separation yields complex I.

(7) Procapsid binding yields complex II which

(8) triggers the ATP-dependent translocation of the packaging proteins along the duplex.

(9) The translocating complex encounters the next cos and again nicks and strand separates the duplex (terminal cos cleavage, downstream nicking reaction). This results in simultaneous release of the DNA-filled capsid and regeneration of complex I.

(10) The addition of a tail, and in some strains tail fibers, yields a fully infectious virus. Details are presented in the text.

​There are two types of lambda cloning vectors

Phagemids

Phagemids = Plasmid + F1M13

Cosmids

Replace this with a sub-header that can be in multiple lines. ​

Yeast Artificial Chromosome

> In 1987, cloning of larger than 45 kb DNA fragments was achieved by the development of yeast artificial chromosomes by D.T. Burke and G.F. Carle.

> The basis of YAC development was that linear eukaryotic chromosomes like commonly used yeast, Saccharomyces cerevisiae requires for its replication not only origin of replications but also centomeres and telomeres.

> The centromeres are required for accurate segregation of chromosomes and the telomeres helps in the safeguard of the integrity of the ends of these linear chromosomes.

> There are three yeast selectable markers genes present Trp 1, Ura 3 and Sup 4.

> e.g. pYAC 3.​

ADVANTAGE OF YAC​

It is useful for cloning very large DNA fragments up to 500 kb.

​

DESADVANTAGES OF YAC

• Very fragile and prone to breakage,

• Unstable, with their foreign DNA inserts often being deleted

• Loss of the entire YAC during mitotic growth

• Difficult to separate the YAC from the other host chromosomes

• The yield of DNA is not high

​

Expression vectors

Heps the target DNA to express inside the host cell...

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells.

The vector is used to introduce a specific gene into a target cell and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene.

Expression vectors are the basic tools in biotechnology for the production of proteins.

The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of a significant amount of stable messenger RNA, which can then be translated into protein.

An example of the use of an expression vector is the production of insulin, which is used for medical treatments of diabetes.​

​Introduction

Elements

1. Origin of replication

2. Selective marker

3. Transcriptional promoter

4. A transcription termination sequence

5. Unique multiple cloning sites

6. The correct translation initiation sequence such as a ribosomal binding site and start codon

7. Translational terminator or a termination codon

8. Protein tags

9. May have elements for transformation or the insertion of DNA into the host chromosome