2.6 recombinant plasmids

features of recombinant plasmids

• origin of replication (ORI)

• antibiotic resistance gene

• multiple cloning site (MCS)

• promoter region

• selectable marker

• screening marker

plasmid

a small circular piece of double-stranded DNA that is able to reproduce independently and may be taken up by cells (usually bacteria) in addition to chromosomal DNA

plasmids naturally exist in bacterial cells

origin of replication (ORI)

section of DNA sequence which is recognised by a cell’s replication proteins, allowing initiation of new DNA synthesis

antibiotic resistance gene

a gene within the bacteria which codes for specific proteins that provides the bacteria with resistance to antibiotics

promoter region

a DNA sequence found upstream of the target gene that acts as a binding site for RNA polymerase

selectable marker

• genes carried by plasmids for certain traits, often for antibiotic resistance

• allows for selection of bacteria that have potentially taken up the recombinant plasmid

screening marker

• also called reporter genes

• typically cause a colour change or other visible change in the tissue of the transformed organism (eg. lacZ)

vector

an agent or vehicle used to transfer pathogens or genes between cells and organisms

making recombinant plasmids

• the DNA of the plasmid and foreign DNA fragments are cut using the same endonuclease, creating complementary sticky ends - changes the plasmid from circular to linear

• reverse transcription often used to create foreign fragments ensuring that non-coding introns are excluded

• foreign DNA fragments and the plasmids are mixed, and, in some cases, their sticky ends pair by weak hydrogen bonds, forming a recombinant plasmid (otherwise plasmids may reseal themselves)

• ligase is added to make the join permanent through covalent bonding - phosphodiester bond

bacterial transformation

process in which bacterial cells take up and express segments of foreign DNA that become part of their genetic makeup

techniques used to increase the uptake of plasmids by bacterial cells:

• electroporation (creates temporary holes in the PM)

• heat shock (increase the fluidity of the PM)

producing human insulin

production of recombinant plasmids

• mature mRNA coding for insulin chains used as a template for reverse transcription

• insulin gene and plasmid cut with same endonuclease forming complementary sticky ends. the two DNA molecules are inserted into two different plasmids using ligase

combining the plasmids and bacteria

• recombinant plasmids mixed with bacterial cells and exposed to heat shock or electroporation, culture is incubated to allow for replication and gene expression

forming the insulin

• bacteria are spread over culture plates containing ampicillin and X-gal to confirm whether the bacteria took up the plasmids and whether the plasmids contain the gene of interest

• the bacteria with the recombinant plasmids undergoes a fermentation process, and are then lysed to extract the expressed protein

• samples are purified, the two chains are mixed together and form disulfide bonds, ingredients added to produce the desired duration of insulin

two different plasmids and bacteria were used, one for insulin chain A and one for insulin chain B

advantages of insulin produced by cloned genes

• high levels of purity

• reliability of supply

• reduced chance of side effects such as allergy (compared to insulin derived from other animals)

• consistency of quality between batches