Plasmids and Vectors Flashcards
(25 cards)
What are some applications of genetic engineering for the production of proteins for human therapy?
1) Purified ‘recombinant’ proteins produced in bacteria, fungal, plant, insect/animal cells
2) Transgenic livestock - production of recombinant protein in milk (e.g. a1-antitrypsin in sheep)
What is an application of genetic engineering in gene therapy?
Correcting symptoms of a genetic disease by introducing the ‘correct’ gene into cells
What is the process of gene therapy using spinal muscular atrophy (SMA) as an example?
1) SMN gene inserted into viral vector
2) SMN gene encapsulated in scAAV9 - virus not integrated into human genome
3) scAAV9 carrying SMN gene delivered to targeted cells/tissues of patient via injection
4) SMN gene introduced to target cells/tissues from virus scAAV9
5) SMN expression restored to normal level in cells which have viral genome
What is the process for mode of delivery of a vector?
1) Vector binds to cell membrane
2) Vector is packaged in vesicle
3) Vesicle breaks down releasing vector
4) Vector injects new gene into nuclease - cell makes protein using new gene
What do transgenics use?
Agrobacterium for plants
What is a vector?
Artificially made molecule that can facilitate incorporation of foreign DNA + manipulation
What is a clone?
Population of cells/organisms derived from a single progenitor - genetically identical
What is transformation?
Process where exogenous DNA is transferred into host cell and replicated
What is gene cloning?
Involves isolation of clones of bacteria expressing sequence of interest (sub-cloning, movement/transfer of DNA)
What is biosynthesis?
Ability of an organism to produce a substance from substrate(s)
What is biotransformation?
Ability of an organism to convert 1 substance into another - usually enzymatic process
What are transgenic organisms?
Organism possessing DNA from another organism which it is able to transcribe
What are the uses of cloning/vectors?
1) DNA vaccines
2) Gene therapy
What are the processes to cloning target DNA>
1) Cut plasmid vector with Aval
2) Excise DNA insert of interest from source using Aval
3) Ligate insert of interest into cut plasmid
What are the steps to ligation?
1) Self-adenylation of DNA ligase
2) Adenyl group transfer to DNA
3) Phosphodiester bond formation
What features can be designed with plasmid vectors?
1) Antibiotic resistance
2) Colorimetric ‘markers’
3) Strong/weak promoters for driving expression of protein
What are 2 methods used to transform bacteria cells?
1) Heat shock
2) Electroporation
What are competent host cells?
Bacterial cells that can take up DNA from environment
How can you increase the likelihood of DNA being taken up in culture?
With an electrical current or divalent cations
What is the process of heat shocking to insert foreign plasmid/ligation product into bacteria?
1) Chemically competent bacteria + DNA
2) Short incubation on ice
3) Heat shock - 42C for 45s
4) Back to ice
5) Growth media added
6) Transformed cells incubated at 37C for 30min with agitation
What is the process of electroporation?
1) Apply electric current - creates temporary ‘pores’ in cell membrane, forcing -ve DNA into cells
2) Carried out at 0C to minimise heat damage to cells - in presence of CaCl2
3) DNA forced into cells by applying 42C heat shock - thermal current sweeps DNA into cells
How can you tell if plasmids were taken up in transformed bacterial cells?
Is grown on selective media (containing an antibiotic) to select for cells that took up a plasmid with resistance gene for that antibiotic
Assess which isolates grew on solid media
What are the conditions that restriciton enzymes have to have specific?
1) Temperature
2) Salt concentration
3) Purity of DNA
Why might a plasmid not be able to generate functional protein from cloned DNA even if it contains the insert?
1) Gene may not be intact/mutations could have been introduced that disrupt it
2) Protein encoded by gene may require post-translational modifications (i.e. glycosylation/cleavage) to function
3) Some enzymes are expressed from separate genes
