PART 2 - Genetic modification - Using enzymes to manipulate DNA – plasmids and insulin and GMOs

Define gene cloning

• The process by which a gene of interest in located and cloned to produce multiple copies

Define recombinant DNA

• DNA that is formed by combining DNA from different sources, often from different kinds of organisms

Define recombinant proteins

• Recombinant DNA in organisms like bacteria or yeast creates proteins. These proteins are made from cloned genes, which carry the instructions for their amino acid sequences.

What are plasmids?

• Small, circular, double-stranded DNA

• Separate from main chromosomal DNA

• They exist naturally in bacterial cells

How are plasmids used?

• Self-replication (contain an origin or replication (ORI) that occurs independently of the main chromosome

• Plasmids that are used to transport foreign DNA into bacterial cells are called vectors

• They can incorporate DNA from any source and then express those genes

What are some advantages of plasmids?

• Antibiotic resistance

Making recombinant plasmid outline

• The DNA of the plasmid is cut using a restriction enzyme (endonuclease) in order to create sticky ends.

• The foreign DNA fragments are prepared using the same endonuclease so that the foreign DNA has sticky ends complementary to the cut plasmid.

  Often, the process of reverse transcriptase is used to create these foreign fragments to ensure that non-coding introns are not included 

• The foreign DNA fragments and the plasmids are mixed

  In some cases, their ‘sticky ends’ pair by using weak hydrogen bonds. A recombinant plasmid has been created. (other pairings will also occur, such as cut plasmids resealing themselves so that they are not recombinant plasmids)

• The joining enzyme, ligase, is added and this makes the joins permanent through covalent bonding

What is transformation?

• Transformation is the process of transferring plasmids into bacterial cells

• Usually uses E.Coli or yeast cells

How can transformation be improved

• Electroporation: Electric field shocks cells, creating holes in the plasma membrane

• Heat Shock: Cells suspended in icy salt solution and then transferred to 42 degrees celsius for <1 minute. This increases fluidity of the membrane

Where was human insulin for medication originally extracted from? What were the side effects?

• The pancreatic tissue of cows and pigs

Side Effects of this include:

• Lower purity

• Lower reliability

• Side effects (due to allergies)

• Less consistent quality

3 step process of producing recombinant insulin (Mrs Macdonald)

1. Human DNA & Plasmid are cut with the same restriction enzyme

2. Insulin gene & plasmids sticky ends match up

3. DNA ligase reforms bonds between DNA

Production of recombinant plasmids in detail (insulin)

• Amino acid sequence of each chain (chain a and chain b) is used to determine DNA sequence

• DNA produced by reverse transcription using mature mRNA strands that code for insulin. Alternatively, DNA synthesisers can be used 

• Insulin gene and plasmid cut with same endonuclease, forming sticky ends. The DNA molecules are inserted into different plasmids using ligase

TRANSFORMATION OCCURS (plasmid introduced to bacteria - where electroporation / heat shock)

• bacteria put onto plate containing ampicillin - eliminates the plate with no plasmid formed

• 3 plates one with no plasmid, one with the plasmid without the gene and one with the plasmid and the gene

• Plate containing tetracycline - the recombinant plasmid is the one with no colonies due to the broken tetracycline resistance gene by human insulin a gene

• Open the found plasmid with insulin gene back up - same restriction enzyme used

• The plasmid contains (adds) the lacZ gene (codes for beta-galactosidase) as a selectable marker 

• (enzyme that breaks down lactose into glucose and galactose) protects against enzymes

• put onto ampicillan again - no plasmid - plasmid with insulin a - plasmid with insulin a and lacZ

• X-gal (made of galactose) - beta-gal if it finds an x-gal molecule, will cut the galactose off and the indole part turns a bright indigo blue - they contain the beta-gal gene

• Chosen plasmid goes through transcription and translation

• In the start of an amino acid chain, an extra code adds a methionine when making insulin A and B genes. This methionine is unique in the sequence. To attach beta-galactosidase to insulin peptide, a chemical treatment breaks the connection between them.

• Cell lysed with lysosomes, allowing the protein to be extracted. The protein is treated with cyanogen bromide, separating the insulin chains from other proteins that were translated 

• The two chains are mixed together and join via disulphide bonds

Other ways to produce insulin

• Proinsulin can be produced by inserting the sequence coding for proinsulin into E.Coli

• The bacteria go through the fermentation process and express genes

• An enzyme is used to modify the proinsulin sequence, resulting in a purified insulin molecule

What are genetically modified organisms (GMOs)?

• Have genomes that have been edited/modified using genetic engineering technology

The organisms could have had:

-A gene or segment of DNA added

-A gene silenced so that its function is lost

What are transgenic organisms?

• Organisms are GMO’s that contain genetic material from a different species

What are transgenes?

• The gene that came from another organism

What is the outcome?

• The desirable characteristics of one organism being expressed in another

Genetically modified organism application in agriculture

• Increase crop productivity – increased photosynthetic and growth rates, greater yields 

• Provide resistance to insect predation

• Prevent disease

Victoria/Australia guidelines about agriculture

• In Australia – insect-resistant cotton crops (1996) – protects against infestation by altering crops to produce their own insecticide. Environmentally friendly

• In Victoria – herbicide-resistant canola crops (2008)

What is needed for the applications in agriculture? What is an example?

• Biological vector needed, due to cell wall 

• Agrobacterium: a vector which naturally infects plants and causes galls because it carries a plasmid with the gene for gall disease

• Modified to be non-infectious and used to transfer a recombinant plasmid

Examples of applications in agriculture

Salt tolerant wheat 

• Salinity a problem in Aust

• Some plants are able to protect themselves by storing built up salt in vacuole or by pumping salt out of their cells

• This gene has been isolated and transferred into wheat plants, improving the yield in salty soils

Bt cotton 

• Crops protected from insects by spraying with insecticides

Insecticides: 

• harmful to beneficial insects and the environment 

• Human health impacted 

• Expensive 

• Bt cotton is a transgenic crop that contains 2 genes from the soil bacterium Bacillus thuringiensis

• These genes make the plant produce a protein which kills the main caterpillar pest

• Over 15 years no adverse effects

Golden rice 

• White rice is low in vitamin A 

• Millions of people rely on white rice as their major food source 

• Transgenic golden rice is produced when 2 plant and 1 bacterial gene are inserted into white rice genome 

• Genes switch on biochemical pathway sending Vit A to the grains rather than the leaves

The regulation of GMOs in Australia

• Office of the Gene Technology Regulator (OGTR) is responsible for regulating GMOs in Australia.

• State governments can further regulate this 

• In 2019, Tas and SA both banned GMOs. This will remain in place in Tas until at least 2029. SA lifted the restrictions in 2020

SOCIAL IMPLICATIONS - Issues arising from GM and transgenic organisms

• Increased food supply, nutritional content and food quality

• Expanded range for growth of agricultural species

• Access to the technology, social equality/inequality 

• Labelling and consumer choice

• Patents and pricing; control of access by biotechnology companies 

• Costs to farmers

BIOLOGICAL IMPLICATIONS - Issues arising from GM and transgenic organisms

• Safety of consuming GMOs

• Cross-pollination between GM plants and wild plants

• Cross pollination between GM and non-GM crops

• Viability of transgenic organisms in the wild

• Health of GMO’s 

• Genetic variation in agriculture

ETHICAL IMPLICATIONS - Issues arising from GM and transgenic organisms

• Violation of animal rights

• Human self-interest overrides ethical treatment of other organisms

• Intervention in evolutionary process

Production of insulin diagram