Transformation

//Transformation

Transformation

Transformation is the process of transfer of exogenous genetic material into the cell through the cell membranes resulting in alteration of genetic material of host cell.

The transformation process required the recipient bacteria must be in a state of competence i.e. state of being able to take up exogenous material from the environment, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient cell.

Types of transformation

  1. Natural transformation
  2. Artificial transformation

Natural transformation:

  • There are many types of cells that  cannot take up DNA efficiently without exposure of chemical or electrical treatment. However, some types of bacteria are naturally transformable ie they can take DNA from environment without requiring special treatment.
  • Bacteria that take up DNA are called competent.
  • It is necessary for the natural transformation that bacteria must be in a state of competence, which happens as a  time-limited response to environmental conditions such as starvation and cell density.
  • Competence for transformation is effected by high cell density and nutritional limitation, conditions associated with the stationary phase of bacterial growth.
  • About 40 species of bacteria which are naturally competent and transformable have been discovered.

Artificial transformation:

  • Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA, by exposing it to conditions that do not normally occur in nature.

Methods of Artificial Transformation:

Transformation in Bacteria

Chemical Method:

  • In this method, for the transformation of gene the cells are incubated in a solution consist of divalent cations i.e. calcium chloride solution, after the incubation it is exposed to a pulse of heat shock.
  • As the surface of bacteria is negatively-charged due to phospholipids and lipo polysaccharides on its cell surface and the DNA is also negatively-charged. The divalent cation of calcium chloride cover the charges by coordinating the phosphate groups and other negative charges, hence allows a DNA molecule to attach to the cell surface.
  • It has been also found that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA.
  • A thermal imbalance is created on either side of the cell membrane due to the exposure of heat-pulse, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

         Electroporation

Electroporation also known as  electropermeabilization, is method of artificial transformation in which an electrical field is applied to cells which leads to  increase the permeability of the cell membrane and make it competence which allow DNA to be introduced into the cell.

  • In this technique, the cells are allowed to shock with an electric field of 10-20 kV/cm which leads to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.
  • Electroporation disrupts the lipid bilayer, leading to efficient molecular transport across the plasma membrane.

Transformation in Yeast

There are various methods has been designed for the transformation in yeast cells

Enzymatic digestion

  • The Yeast cells are treated with enzymes to enhance the transformation. The enzymes helps to degrade their cell walls which leads to production of spheroplasts. These cells are very fragile but take up foreign DNA at a high rate.
  • By the exposure of spheroplasts of yeast cell to alkali cations such as those of cesium or lithium leads the cells to take up plasmid DNA. 
  • Other way of transformation with this method also includes the use of  lithium acetate, polyethylene glycol, and single-stranded DNA.  In this process, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation.

Transformation in Plants:

The process of transformation in plants involves the insertion of  DNA from any organism is into the genome of a species of interest. The inserted DNA is called a “transgene”, and the plant obtained after the insertion of trans gene is called “transgenic plant”.

Various methods are available for transfer of DNA into plant cells.

Agrobacterium-mediated transformation:

The most  easiest and most simple way of transformation in plant is  Agrobacterium-mediated transformation.

  • Agrobacterium tumefaciens is a bacteria which is widely found in soil and causes crown gall, and it also has the ability to introduce new genetic material into the plant cell.
  • The genetic material that is incorporated is called T DNA i.e. transferred DNA which is located on a Ti plasmid. A Ti plasmid is a circular DNA found in almost all bacteria.
  • This ability  of bacteria to alter the plant’s genetic makeup was leads to design the plant transformation using Agrobacterium.
  • Currently, Agrobacterium-mediated transformation is the most commonly used method for plant genetic engineering because of relatively high efficiency. Initially it was believed that this Agrobacterium only infects dicotyledonous plants, but it was later established that it can also be used for transformation of monocotyledonous plants such as rice.
  • Several components of the Ti plasmid plays as important role in effective transfer of the genes of interest into the plant cells. These include:
  • T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred into the plant genome
  • vir genes (virulence genes), required for transferring the T-DNA region to the plant but are not themselves transferred, and
  • modified T-DNA region where the genes that cause crown gall formation are replaced with the genes of interest.

Agrobacterium-mediated Plant Transformation Process

The Agrobacterium-mediated transformation process involves a number of steps: (a) isolation of the genes of interest from the source organism; (b) development of a functional transgenic construct including the gene of interest; promoters to drive expression; codon modification and marker genes to facilitate tracking of the introduced genes in the host plant; (c) insertion of the transgene into the Ti-plasmid; (d) introduction of the T-DNA-containing-plasmid into Agrobacterium; (e) mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into plant chromosome; (f) regeneration of the transformed cells into genetically modified (GM) plants; and (g) testing for trait performance or transgene expression at lab, greenhouse and field level.

By |2018-04-19T09:13:10+00:00April 19th, 2018|Molecular Biology|Comments Off on Transformation

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