Vectors for Gene Cloning: Plasmid (Part-1)

A vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector is needed to allow replication of the desired gene in the host organism. Also, foreign DNA in native form is prone to attack by nucleases present in the host organisms.

1)      Cloning Vector

2)      Expression Vector

                                                            

ü  Cloning Vector

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher an organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Allow the exogenous DNA to be inserted, stored, and manipulated mainly at the DNA level.

 

ü  Shuttle Vector

A shuttle vector is a vector constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types.

It has two origins of replication, each of which is specific to a host. Since shuttle vectors replicate in two different hosts, they are often known as bifunctional vectors.

One of the most common type of shuttle vector is the Yeast Shuttle Vector. Almost all commonly used Saccharomyces cerevisiae vectors are shuttle vectors. Yeast shuttle vector has components that allow for replication and selection in both E.coli and Yeast cells. The E.coli component of a yeast shuttle vector includes an origin of replication and a selectable marker, e.g. antibiotic resistance, beta-lactamase. The yeast component of the yeast shuttle vector includes Autonomously Replicating Sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker (e.g., URA3, a gene that encodes an enzyme for uracil synthesis).

Example of Shuttle Vector: pHV14, pEB10, pHP3, etc. replicated both in Bacillus subtilis and E.coli.

pJDB219 is another shuttle vector that can replicate in E.coli and Yeast. pJDB219 is another shuttle vector that can replicate in E.coli and Yeast (Saccharomyces cerevisiae).

 

Essential characteristics of  Vectors

·         Should have antibiotic resistance genes used for selection of bacterial hosts (selectable marker).

·         Most plasmids have at least one DNA sequence that can act as the origin of replication (ori)- help in independent multiplication.

·         Multiple Cloning Sites (MCS) for cleavage by more than one type of restriction endonucleases.

 

Different types of Vectors

1)      Plasmid Vector

2)      Phage Vector

3)      Phagemid / Phasmid

4)      Cosmid

5)      Bacterial Artificial Chromosome (BAC)

6)      Yeast Artificial Chromosome (YAC), etc.

 

 

ü  Plasmids

First cloning vectors, circular molecules of dsDNA, have an independent existence in the bacterial cells. Easy to isolate, purify, and reintroduce into a bacterium by the transformation. Some plasmid replicates by inserting themselves into the bacterial chromosome (integrative plasmid/ episome).

 

Features:

1)      Size and Copy number

Less than 10 kb is desirable for a cloning vector. Copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell.

·         Stringent Plasmid- larger plasmid, low copy number (1-2 per cell)

·         Relaxed Plasmid- multiple copies of 50 or more per cell.

Generally a useful cloning vector needs to be present in  the cell in multiple copies to obtain a large copy of recombinant DNA molecule.

 

2)      Conjugation and Compatibility

Conjugation plasmid has the ability to promote sexual conjugation between bacterial cells. Conjugation and plasmid transfer is controlled by tra genes. Compatibility is the ability of different plasmids to coexist in a cell. Different plasmids must be compatible for successful gene cloning experiments to prevent loss of cloned DNA.

 

Classification of Plasmids

·         Fertility or F plasmids carry tra genes, F plasmid of E. coli.

·         Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin, and mercury.  RP4 of Pseudomonas.

·         Col plasmids code for colicins, ColE1 of E. coli.

·         Degradative plasmids allow the synthesis of unusual molecules such as toluene, salicylic acid. TOL plasmid of Pseudomonas putida.

·         Virulence plasmids confer pathogenicity to host bacterium. Ti plasmid of Agrobacterium tumefaciens.

Advantages and Disadvantages

ü  Advantages

·         Small, easy to handle.

·         Straight forward selection strategies.

·         Useful for cloning small DNA fragments (less than 10 kbp)

ü  Disadvantages

·         Less useful for cloning large DNA fragments (larger than 10 kbp)

 

Ti plasmid of Agrobactgerium tumefaciens- plant vector

Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing or Ti plasmid to susceptible plant cell- crown gall disease. Ti plasmid ranges from 180-205 kb in size. It has T DNA which is of  approx 20 kb and several genes such as vir genes for virulence, ori gene for the origin of replication, tra genes for transfer and genes for opine synthesis. Virulence genes are responsible for the transfer of T DNA  into the host cell and integration of T DNA  with the host genome. Different strategies are used to introduce the desired gene in to plant cells.

The Ti plasmid as a vector in plant genetic engineering