Biotechnology

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The main application of in vitro genetic manipulation is that it has:

Extended the range of products that may be produced by microorganisms

Provided new methods for increasing the yields of existing products

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Plasmids:

Circular molecules of DNA.

Extrachromosomal genetic element, widespread in bacteria.

Lead independent existence within the bacterial cell.

Copy number can vary around 1 to 50.

Compact due to supercoiling.

Carry genes for antibiotic resistance

Plasmids can be spread between bacteria, sometimes between different species.

Many different types of plasmids, vary widely in modes of replication, transfer, modes of action.

Plasmids as vectors:

Plasmid vectors allow specific DNA to be rapidly grown up in large numbers

Plasmids can then be re-isolated from the bacteria. • Vectors commonly used as a high quality template for DNA sequencing.

DNA and plasmids are digested with the same enzyme.

DNA is added to the digested plasmid and DNA ligase is added.

Plasmids inserted into the cell (transformation into competent cells). • Cells grown in the presence of the selection agent.

Plasmid carries the gene for antibiotic resistance.

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Restriction Enzymes:

Also known as restriction endonucleases

Responsible for the phenomenon in bacteria known as hostcontrolled restriction modification. These enzymes are able to cleave DNA from other strains but not that of the original strains.

These restriction enzymes cleaved DNA at a specific recognition site.

Name = bacterial origin e.g. Escherichia coli = Eco. The strain or type identification follows without italics: e.g. EcoR.

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Expression Vectors:

Expression systems are used for the artificial synthesis of proteins encoded by recombinant-DNA constructs. This is useful for several reasons:

The production of biotechnologically relevant proteins (enzymes, pharmaceuticals).

The selection of clones via the specific detection of a gene product, e.g. by immunoscreening.

The analysis of interactions of the gene product with other molecules by testing expression products of gene fragments or by introducing mutations (e.g., epitope mapping).

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Subunit Vaccines:

A subunit vaccine presents an antigen to the immune system without introducing viral particles.

One method of production involves isolation of a specific protein from a virus and administering this by itself. A weakness of this technique is that isolated proteins can be denatured and will then become associated with antibodies different from the desired antibodies.

A second method of making a subunit vaccine involves putting an antigen's gene from the targeted virus or bacterium into another virus (virus vector), yeast (yeast vector) or attenuated bacterium (bacterial vector) to make a recombinant virus or bacteria to serve as the important component of a recombinant vaccine (called a recombinant subunit vaccine).

The recombinant vector that is genomically modified will express the antigen.

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Vaccine Summary

Recombinant vaccines:

Delete the virulence genes (live vaccine that is non-virulent -attenuated)

Non pathogenic carriers with genetically engineered antigenic structures (vector vaccines)

Antigenic proteins cloned, expressed and used as protein (subunit vaccine)

DNA vaccines:

Inject with genetically engineered DNA (directs cell to produce antigenic substance. West Nile virus - horse vaccine was one of the first.

Subunit vaccines:

A subunit vaccine is a fragment of a pathogen, typically a surface protein, that is used to trigger an immune response and stimulate acquired immunity against the pathogen from which it is derived.

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Gene Therapy

Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene.

Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell.

Insert working copy of a gene into cells to treat disease. • Repair faulty gene • Express something which will be beneficial w.r.t. a disease

Delivery challenges • Viruses - retroviruses • Stem cells • Coated nanoparticles

Triggering oncogenes – depends where in the chromosome the new gene is incorporated.

Still many challenges – new genes must be controlled by the body.

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RNA interference (RNAI):

Gene silencing/knockdown

Small pieces of double stranded RNA

Small interfering RNA (siRNA): Suppress expression of genes with sequence homology

Research: understand function of genes by silencing them

Treatment: Silence disease causing genes, Neutralise infecting agents

Manipulation of iRNA:

Synthetic iRNA: Small molecule inhibitor of any gene of choice. Used for Chronic viral infections eg. Hep C, HIV, Neurodegenerative diseases: Dominant mutant allele, Treating acute infections, Silencing proinflammatory genes to prevent tissue damage, Controlling cancer by silencing regulators involved or oncogenes

Challenges: Delivery and survival Selecting unexpected target Displacement of natural role in cell

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CRISPR or CRISPR-Cas9:

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea.

Hence these sequences play a key role in the antiviral defense system of prokaryotes.

Edit DNA at precise locations.