20 Background: Plasmid Isolation and Bacterial Transformation
Genetic information in bacterial cells
Prokaryotic cells differ from eukaryotic cells in several ways (Table 1). Both prokaryotic and eukaryotic cells store and transmit genetic information in the form of DNA molecules. Most of the genetic information in a bacterial cell is part of one circular DNA molecule that is typically between 1 x 106 and 1 x 107 base pairs (bp) in length. Many prokaryotic cells also contain small circular plasmids that replicate independently of the main chromosome and carry a small number of genes. Some of these plasmids replicate independently of the cell cycle and hence can be present in hundreds of copies per cell. This simplifies the purification of these plasmids from bacterial cells and the large circular chromosome.
|
|
prokaryotic |
eukaryotic |
|
Typical cell size |
smaller |
larger |
|
Nucleus and membrane-enclosed organelles |
absent |
present |
|
Molecule that stores and transmits Genetic information |
DNA |
DNA |
|
Type(s) of cell division |
Binary fission |
Mitosis and meiosis |
|
Organization of genome |
one circular chromosome/ small circular plasmids |
Multiple linear chromosomes in nucleus, circular DNA in mitochondria and chloroplasts |
Table 1: notable differences between prokaryotic and eukaryotic cells.
One can choose between numerous kits and procedures to isolate plasmid DNA from bacterial cultures. The basic outline of procedures that involve a DNA binding column is as follows:
- Grow the bacterial culture in a liquid growth medium containing the antibiotic that serves as the selectable marker for the plasmid of interest. Bacterial cells lacking the plasmid are thus being selected against by the antibiotic improving plasmid yield.
- Use a centrifuge to spin down the dense bacteria to form a pellet. This allows the scientist to remove the growth medium (supernatant) from the bacterial pellet.
- Disperse the bacterial pellet in a buffered solution (solution 1) such that subsequent chemicals have greater access to the cells.
- Add a denaturing agent (solution 2) to disrupt cell membranes allowing DNA to exit cells. This could come in the form of a detergent or high pH. The tube contents are mixed gently to reduce breakage of chromosomal DNA.
- Add a neutralizing agent (solution 3) and mix gently. Much of the cell debris will have precipitated by this point; however, the plasmid DNA remains soluble in the supernatant.
- Spin the tubes in a centrifuge to separate insoluble debris from the supernatant. Transfer the supernatant to a DNA binding column.
- Spin the supernatant through the DNA binding column. The DNA will bind but liquid will pass through.
- Wash the column to remove impurities from the DNA. Spin tube in a centrifuge to move the wash solution through the column leaving the DNA on the column matrix.
- Elute the DNA from the column using a small volume of low salt buffer. The DNA can now be used in downstream applications such as agarose gel electrophoresis, bacterial transformation, restriction enzyme digestion, or DNA sequencing.
Procedures that do not rely on a DNA purification column involve heat, detergent, or base to lyse the cells so the DNA can exit the cell. Ethanol or isopropanol is then used to precipitate the DNA to purify and concentrate it.
Bacterial transformation
Some types of bacteria are naturally competent to acquire DNA from their environment. In 1928, Frederick Griffith showed that Streptococcus pneumoniae could acquire new traits by taking up a “transforming principle” from heat-killed cells. Transformation of many types of bacteria happens very infrequently unless they have first been treated in a special way. For instance, E. coli cells can be made competent for transformation by pre-incubating them in a cold Ca+2 or Mn+2 solution.
Transformation of E. coli with plasmids tends to be inefficient even after the cells have been made competent. Plasmids are circular DNA molecules that are special in that they include an origin of DNA replication (allows production of many copies of the plasmid), a selectable marker (allows only transformed bacteria to divide and produce new cells), and a mechanism by which the DNA of interest can be introduced (Figure 1). It is important to realize that it is not the plasmid itself that allows the transformed cells to grow, but growth is possible because a gene on the plasmid is expressed in the cell. Therefore, the transformed cells are incubated for 15 to 30 minutes after transformation in liquid growth medium lacking the selective agent prior to being plated on solid growth medium that includes the selective agent (such as the antibiotic ampicillin). Overnight incubation at 37oC will allow transformed cells to grow and divide to forma colony.
Figure 1A: special features of a plasmid and B: outline of the transformation procedure.
If a gene of interest is introduced into the multiple cloning region in conjunction with a bacterial promoter, the encoded protein can be translated in the transformed cells. The researcher can then analyze the production of that protein in the colony. In this lab, we will be looking at the expression of the Green fluorescent protein and some of its derivatives. We can analyze the expression of these proteins by shining ultra-violet light; each protein will fluoresce at a particular wavelength, and hence the colony will appear to be a particular color.