Recombinant DNA Flashcards
Vectors
A type of recombinant tool that can be used to transfer DNA into a biological system like a bacterium or cell.
4 types of pf vectors
- Plasmids
- Bacteriophages
- Viruses
- Or artificial chromosomes
Plasmids
- found in many but not all bacteria
- Generally Have a restricted host range
- Are transferable by various means including transformation and conjugation
Phages
- Lambda – bacterial viruses
* transfer of antimicrobial resistance through a mechanism called transduction
What are vectors used for?
Used to mutate a gene and understand the functional parts of a protein or the effects of a specific mutations on proteins structure or function
Plasmid features
- Can be linearized at one or more sites in non-essential stretches of DNA
- Can have DNA inserted into them
- Can be re-circularised without loss of the ability to replicate
- Are often modified to replicate at high multiplicity (copy number) within a host cell
- Contain selectable markers
- Most are relatively small 4-5kb in size
Why is the selection of vectors important?
have the correct features for inserting the gene selecting for recombinants and may also require promotors or other elements in the correct place.
Vectors, PCR, restriction enzymes and DNA ligase
- The vector and the PCR amplicon of the gene must be cut with restriction enzymes to produce compatible ends
- These are then joined by ligation using a DNA ligase
Recombinant Proteins
Having made the recombinant plasmid we can artificially transduce bacteria where the plasmids will replicate and be maintained in the presence of a selectable marker such as ampicillin.
Clones and recombinant proteins
purify the protein produced, investigate its properties or function,
• Or alternatively develop and produce therapeutics
Why use plasmids as recombinant tools
• Expression of a recombinant gene in a living organism of choice
- Prokaryote or eukaryote
• Add or modify control elements
- Make it inducible or express it to high levels on demand
• Alter the properties of the gene product
- Make it secreted extra-cellularly or into the periplasmic space
- Fuse it to a peptide tag or other protein
- Make I useful as a therapeutic
Recombinant vectors and real world usage
- Human insulin - diabetes
- Interferons-a & b – viral Hepatitis or MS
- Erythropoietin – kidney disease, anaemia
- Factor XIII – haemophilia
- Tissue plasminogen activator (TPA) – embolism, stroke
Biologics
- First appeared in the clinic in the late 1980s
- around 50% of the 62 recombinant drugs approved by the FDA for clinical use between 2011 and 2016
- 17 biologics were approved in 2017, double the previous average
Synagis
Respiratory Syncytial Virus -
Humanised anti-RSV IgG1
Herceptin
HER - 2 positive breast cancer - Humanised anti-HER -2 IgG1
