Applied Biology - Class 12
- DNA cloning and recombinant DNA
- A brief history of restriction enzymes
- Restriction enzyme mechanism
- Parts of a cloning vector
- Features of cloning vectors
- Multiple cloning sites & restriction enzymes
- Insertional inactivation (two antibiotic selectable markers)
- Insertional inactivation using Lac Z gene (Blue white screening)
- Competent cells, transformation, and other methods of DNA delivery
- Cloning sites and insertional inactivation
How to make hosts take up foreign DNA
|plasmid||circular DNA found in bacteria; can replicate independent of chromosomal DNA|
|competent cells||cells with permeable membranes that can readily take up DNA|
|transformation||the process of forcing competent cells to take up foreign DNA|
|heat shock||exposure of competent cells to raised temperatures to increase chances of taking up foreign DNA|
How do you insert foreign DNA into a host organism?
Once a piece of foreign DNA has been successfully ligated into a cloning vector, the recombinant plasmid is inserted into host organisms like bacteria (or other organisms like yeast). The image below illustrates the addition of a plasmid to the cytoplasm of an E.coli bacterium.
As you might imagine, these hosts need to take up the plasmids through pores in their cell membranes. This brings us to the next question - how do you pass negatively charged DNA through a cell membrane?
The answer is - scientists use what are known as competent cells, which are much have highly porous membranes, and are thus better at taking up foreign DNA.
The next section explains what make these cells special, using bacteria as examples.
Competency and transformation
DNA is a hydrophillic molecule, which means it likes to mix with water. However, bacteria have cell membranes that are full of lipids - which make it very hard for DNA to pass through them and into the cell. Competent cells are those whose membranes have been altered to make them more porous.
Chemical competence is commonly brought about by incubating bacteria on ice in a solution containing calcium chloride. The positive charge of the calcium ions neutralizes the negative charges on the bacterial lipid bilayer and DNA.
Next, the bacteria are treated to a "heat shock" - typically, they are briefly subjected to
degrees C, before replacing them on ice. This step also helps make the bacteria more porous to external DNA, and increases the chance of successful uptake of the plasmid.
This process of forcing foreign DNA into a host is known as transformation.
Other techniques to deliver DNA to cells
- Microinjection is a method that allows DNA to literally be directly injected into the nucleus of a cell.
- Biolistics is another method that is used commonly in plant cells. Micro-particles of gold or tungsten are mixed with DNA molecules, and then 'shot' at cells at high velocities using a 'gene gun'.
- Diasarmed pathogens, like the Agrobacterium tumifaciens bacterium, or several retroviruses, can be used to deliver DNA safely to cells without causing any harm to them.
test your understanding 1
Which of the following options best describes the process of transformation?
test your understanding 2
Why do some bacteria need to be made competent before they can be used for transformation?