4E PP - Bacterial Recombination & Transformation

Genetically Modifying Bacteria

Scientists can insert foreign DNA into bacterial plasmids, that are then be taken up by bacteria.

The bacteria are then made to express the gene, producing a desired protein.

Plasmids

Plasmids are small loops of DNA that replicate independently from the chromosome.

They vary in length but can be up to 200kb long.

Plasmids contain a small number of genes, but the most useful relate to antibiotic resistance.

Have a

• Promoter to drive target gene expression

• Target gene

• Antibiotic resistance gene

Gene Cloning

Gene cloning is an alternative to PCR used to generate many copies of a DNA sequence.

This is performed by inserting a gene of interest into bacteria, via plasmids.

Gene cloning is often preferred to PCR because bacteria also create the protein associated with the gene of interest.

Recombination

A plasmid can be edited to incorporate a gene of interest.

Once the gene is inserted, the plasmid is referred to as a recombinant plasmid.

Making Recombinant Plasmids

To create recombinant plasmids, the following are required:

1.Gene of interest

2.Plasmid vector

3.Restriction endonuclease/s

4.DNA ligase

Gene of Interest

The gene of interest is the sequence of DNA that codes for the protein scientists wish to synthesise.

The gene of interest is isolated and amplified using PCR.

Plasmid Vectors

A plasmid vector is a means of introducing foreign DNA into an organism

Most plasmid vectors contain:

1.Known endonuclease recognition sites

2.Antibiotic resistance gene/s e.g. ampicillin, tetracycline

3.Origin of replication or ORI (signals start site for DNA replication)

4.Reporter gene

Reporter Genes

A reporter gene has an identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest.

Restriction Endonucleases

The gene of interest and the plasmid are both cut with the same endonuclease to generate complementary sticky ends.

The cuts are made to disrupt an antibiotic resistance or reporter gene.

Note: blunt end endonucleases may also be used but are less specific, as a blunt end can bond with any other blunt end.

DNA Ligase

DNA ligase repairs the phosphodiester bonds between the gene of interest and the plasmid vector.

Recombinant Plasmids

Plasmids will not always take up the gene of interest, resulting in a mixture of non-recombinant and recombinant plasmids.

Bacterial Transformation

Bacteria can be made to take up recombinant plasmids; a process called bacterial transformation.

Bacteria can then be used to synthesise specific proteins coded for by the gene of interest.

Uptake of Recombinant Plasmids

There are 2 primary methods of promoting uptake of recombinant plasmids by bacteria:

•Heat shock

•Electroporation

Heat Shock

• Bacteria & plasmids placed in calcium ion solution to make plasma membrane more permeable.

• Solution is heated to 37-42℃ for 25-45 seconds

• Solution is chilled

• Sudden change in temperature causes the plasma membrane to become more permeable, allowing plasmid vectors to enter the cytoplasm of bacteria.

Electroporation

• Bacteria & plasmids placed in solution to make plasma membrane more permeable.

• Solution exposed to electric current

• Current causes plasma membrane to become more permeable, allowing plasmid vectors to enter the cytoplasm of bacteria.

Antibiotic Selection

To distinguish between transformed and untransformed bacteria, the mixture is exposed to an antibiotic on an agar plate, based on the antibiotic resistance gene in the plasmid.

Only the transformed bacteria will contain plasmids with the antibiotic resistance gene; all untransformed bacteria will die.

Reporter Genes

Transformed bacteria may take up a non-recombinant plasmid and/or recombinant plasmid.

In bacteria with non-recombinant plasmids, the reporter gene will be expressed and show the characteristic phenotype.

In bacteria with recombinant plasmids, the reporter gene will not be expressed, and hence, show no associated phenotype.

Protein Production & Extraction

Transformed bacteria are cultured (grown) and produce the target protein from the gene of interest.

The protein is extracted and purified from other proteins the bacteria produces.