INSERTING A GENE INTO A PLASMID VECTOR

                          WHAT IS A VECTOR?

A vector is a small circular piece of DNA used to artificially carry genetic material into another cell.

For example; plasmids ,viruses ,liposomes

Plasmids are most commonly used as vectors.

Plasmids are normally found in bacterial cells and often contain genes for antibiotic resistance and can be exchanged between bacteria of the same species or of different species through bacterial conjugation.

Bacterial Conjugation is the transfer of genetic material through direct cell to cell contact or by a bridge-like connection between two cells

Why are plasmids used in gene cloning?

Plasmids have properties that allow them to be used for this process. They are listed below:

An Origin Of Replication- This allows foreign (desirable) DNA to be replicated within the host cells.

Marker Genes- This allows identification of cells that have taken up the plasmid

Several single target sites for different restriction enzymes

Low Molecular Mass- This is so that it is taken up easily by bacteria

                           How are Plasmids used as vectors?

A piece of DNA is inserted into the plasmid and the plasmid is used to take the DNA into the bacterial cell.

The bacteria containing the plasmids are treated with enzymes to break down their cell walls

The naked bacteria is then spun at high speed in a centrifuge to separate the larger bacterial chromosomes from the much smaller plasmids

The circular DNA of the plasmid is cut open using a restriction enzyme and the same enzyme is used to cut the gene that should be used so that the sticky ends will be complementary.

 The sticky ends of both the gene and the plasmid DNA are linked together using Ligase Enzyme.

The end product is called Recombinant DNA

Recombinant DNA is DNA made by joining pieces of DNA from two or more sources/species

Getting Plasmids Into Bacteria

The bacteria is first treated by putting them into a highly concentrated solution of Calcium ions.

It is then cooled and given a heat shock to increase the chances of plasmids passing through the cell surface membrane

Only a small proportion of the bacteria actually take up the plasmids and when this occurs it is called Transformation.

Identifying bacteria with recombinant DNA

To identify which bacteria have been transformed successfully, they are spread on agar plates containing an antibiotic.

The bacteria with the recombinant DNA would be able to grow on the agar containing the antibiotic. This is because the plasmid with the recombinant DNA may contain genes for resistance to that antibiotic.

The DNA polymerase in the bacteria with the recombinant DNA copies the plasmids and divides by binary fission so that each daughter cell has copies of the gene. The bacteria then transcribe the new gene and translates it to give the required gene product. For example; insulin

Synthesizing Genes For Insulin

Sometimes locating and isolating the gene for human insulin can be difficult.

So instead of cutting out the gene, genetic engineers extract the mRNA which codes for insulin from the Beta cells in the pancreas and incubate it with the enzyme Reverse Transcriptase.

This enzyme reverses transcription by using mRNA as a template to make single-stranded DNA molecules which are later converted to double-stranded DNA molecules by using DNA polymerase to assemble nucleotides to form the complementary strand.

This is then inserted into a plasmid to transform a bacterium.