dna Logo The Molecular Biology Notebook Online
A Beginners' Guide to Molecular Biology



You are here: Biotechnology > Gene Cloning > Transformation

Cell transformation

The act of introducing a foreign DNA into a cell is called transformationask Dr Chromo!. Cells which are commonly used for transformation are from E. coli, or other bacteria. Other systems, like mammalian or insect cells, may be used.

In bacteria, the DNA introduced is often a plasmidask Dr Chromo!. When the DNA is a bacteriophageask Dr Chromo!, or phageask Dr Chromo!, the process is different. It is called transfectionask Dr Chromo!.

Any normal bacterial cell cannot immediately be transformed. It needs to be made 'Competentask Dr Chromo!' first. A competent cell is one which has the ability to be transformed. A cell becomes competent when it has been specially treated. Cells from a culture in an exponential or log phase of growth are more easily made competent than cells in other stages of growth.

      How to make competent cells

      How to transform bacterial cells with foreign DNA

      Controls used in a transformation experiment

How to make competent cells

A competent cell is a cell that has become fragile, and therefore will be able to incorporate DNA. There are several ways to make a cell fragile.

All methods to make competent cells use cells which are at an exponential phase of growth. Cells from a culture in an exponential or log phase of growth are more easily made competent than cells in other stages of growth. In order to be sure the cells are in such a phase, a small colony is picked out and then a chosen medium inoculated with it. By the next day, the culture will have grown. A small volume is taken out of this overnight culture and poured into a new medium. This is then put in an incubator for three hours.This produces cells that are ready to undergo the rest of the procedure.

These procedures aim at creating tiny holes in the outer wall of the membrane, ensuring the plasmid (recombinant) DNA will have closer access to the cell and will be able to penetrate it. Often glycerol, or another antifreeze agent is added when the cells need to be preserved in the deep freezer. Usually, a large batch of cells is prepared and some of it is kept under refrigeration for later use.

How to transform bacterial cells with foreign DNA

The following is an example of a method to transform competent cell with a plasmid.

Once the cells are made competent, it is very easy to transform them with foreign recombinant DNA. DNA and bacteria are kept on ice for half an hour to enable the DNA to diffuse gently amongst the cells. Then the tube in which the cells are kept is put at 42°C for thirty seconds (this time can be adjusted depending on the size of the tube) and then back to 37°C. This action is called the 'Heat shock'. It provokes a reaction in the cells which will incorporate the DNA which is at their surface. The cells are then kept at 37°C for another 30 minutes, or enough time to allow the expression of resistance to antibiotic. They are then plated onto a medium containing the appropriate components (Inducers? Antibiotics? Substrates?). After 18 hours, transformed bacteria would have given rise to colonies which can be counted and analysed.

Again as with making competent cells, the details of the methods used to transform bacteria varies between different laboratories. Here are a few.

1) This one gives an introduction on competent cells, and a brief method for transformation
2) From the Soars Lab Web server a method to transform E. Coli
3) From the Channing Laboratory : a description of making competent cells and transforming them.

Controls used in a transformation experiment

You might need to understand the concept of a control experiment before reading this chapter.

At each step of this quite long process, something can go wrong. There is a way to find out if it has.

We need additional controls for the transformation

Sample DNA Controls what?
Control 1: A known amount of plasmid Control 1: We will obtain quite a lot of colonies after this transformation. The number of colonies compared with the number of plasmids (or ug of DNA) used in the transformation will give an idea of the efficiency of transformation and ensures transformation has worked. Ligation may not have, so this will let us know.
Control 2: No DNA or some water Control 2: Are the bacteria used all suceptible to our antibiotic (Ampicillin)? We would expect no colonies on that plate and convincees us that the colonies obtained on other plates are due to the correct transformation of a plasmid.
Control 3: Three different densities of our sample ligation Control 3: This is true as well for the control 2. We are never sure of how well or badly the transformation has gone. If the transformation has gone too well, a lot of clones will grow on the plate. and it will be covered in colonies: we won't be able to separate them for further use. It is good to have three different dilutions of the sample to be sure there will be at least one plate where clones will be separated. Here again, it is a matter of trial and error.

How would a cloning experiment take place?

One wants to insert a piece of DNA into a plasmid which carries resistance to Ampicilin, and a gene for the blue / white test . This plasmid is digested with two restriction enzymes EcoRI and BamHI. The sample DNA is also digested with these two enzymes and a fragment of 7kb is located on a gel: this is our sample insert. The slice of gel containing this fragment is cut out, and the sample insert DNA extracted.

Controls to perform during a ligation experiment:

Each control reaction of the ligation creates a sample which will be used to transform a batch of bacteria.

Vector Insert Ligase
Yes/no		 To check?

Digested plasmid

none

no

 1) Is digestion of the vector by the restriction enzyme complete?

Digested plasmid

none

yes

2) Is double digestion working? Can the plasmid self-ligate?

Digested plasmid

Small insert: test fragment

yes

 3) Is the ligase working properly?

The ligations are therefore set up as follow

All the volumes are made up to 50µl

After 16 hours at 16°C, 100 µl of competent cells are ready to be transformed with the ligase reaction products, and other controls. The transformations are set up as follow: each tube contains a certain volume (usually 100µl) of competent cell. The following reaction products are added in each tube.

After the process of transformation (Heat-shock in some cases), each sample is plated onto agarose containing ampicillin, X gal, and the inducer IPTG.


After keeping over-night at 37°C, the plates should look like this:

How well do you think this went?

Can you calculate an efficiency of transformation?

Why do you think you obtain the small number of colonies in the plate 8?

Which are the colonies that contain a plasmid with an insert?

Exploitation of the results

There are plenty of white colonies on plate 8, and they are well separated, even at the high density (Plate 8). If you count the colonies in plate 6, and compare with the total amount of plasmid you put in the transformation, you could have an idea of how well the competent cells work.

The colonies which contain a plasmid with an insert are the white ones found on plate 8, 9, and 10. It is best to pick them from a plate where the colonies are well separated.

An additional note: if the plates are left too long in the incubator, the protein which gives resistance to antibiotics will start leaking out of the colonies (white or blue). This will enable other bacteria present on the plate to grow around the white colonies. These small colonies, which are called sometimes "opportunist" or "satellite colonies", are white and are just the result of the multiplications of some of the bacteria which were not transformed, and were only dormant under the influence of the antibiotic.

An additional exercise is proposed here

Copyright Rothamsted Research 1999- 2010
Web technical : Nathalie Castells-Brooke