Plasmids and Transformation

The method we are mainly concerned with for the project is biological transformation, using 'plasmids' or 'vectors' as they are known in Genetic Engineering.

Plasmids
"Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used."

source: http://en.wikipedia.org/wiki/Plasmid

Transformation
Transformation is the process of inserting exogenous DNA material into a cell. For our purposes, this means the uptake of the plasmid - containing the desired foreign DNA - into the host cell.

The following diagram illustrates the whole process.

source: http://www.accessexcellence.org/RC/VL/GG/plasmid.php

Here, we see that the desired gene is cut from the foreign DNA using a restriction enyzme. The plasmid is similarly cut. This produces 'Sticky Ends', with the ends of the genes not having matching bases. A DNA Ligase is then used to join the ends of the gene of interest with the plasmid. This produces the recombinant DNA.

The plasmids are then inserted into the host cells. To make sure we only have cells that contain the gene of interest, we introduce an anti-bacterial mixture. The plasmids with the foreign DNA have been given a marker that protects against the anti-bacterial, preserving all the cells which contain the foreign gene.