How to clone a gene and express a drug from a bacteria Flashcards
Isolating a target gene
Harvest a copy using restriction enzymes
Amplify a copy using PCR
Synthetically produce a copy
Isolating using restriction enzymes
Cut out the gene of interest
Purify the product
Glue into a vector
Restriction endonucleases: a means of cutting DNA
Enzymes isolated from bacterial cells Provide a protective mechanism against foreign DNA Degrade the DNA of invading viruses Recognise specific DNA sequences Usually 4-8 bp
Use of gel electrophoresis to separate DNA
Separate DNA fragments by their size Apply an electric field DNA is negatively charged Separate through gel matrix Visualise DNA fragments
Cloning vectors
Most simple is a bacterial plasmid
A circular piece of replicating DNA
Range in size from 1000-200K base pairs
May express antibiotic resistance gene or other survival genes
Can be modified to express genes of interest
DNA ligase
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’ hydroxyl ends of one nucleotide with 5’ phosphate ends of another. ATP is required for the ligase reaction.
How to insert the gene into the host and isolate the cloned gene
Double-stranded recombinant plasmid DNA introduced into bacterial cell
Isolate bacteria containing target DNA
Propagate colonies of interest
Cell culture produces hundreds of millions of new bacteria
Characterisation- many copies of purified plasmid isolated from lysed bacteria
Introduction of vector into host cell
Different approaches for each host
Bacteria: CaP/ heat shock, electroporation
Yeast: electroporation, DMSO
Mammalian cells: CaP, electroporation, lipofectin
Bacterial transformation
DNA molecules are too large to easily diffuse or be transported through the cell membrane
Use: natural competence, calcium chloride/ heat shock- competent cells uptake DNA after heat shock, electroporation- electrical shock makes cell membranes permeable to DNA
Lipofectamine mediated transport of DNA into eukaryotic cells
Induced membrane fusion between vesicles and plasma membrane of target cell releases substance into the cytoplasm
Selection of clones containing the desired gene
Antibiotic resistance
Nutrient utilisation- colour change
Screen for presence of the gene
Screen for expression of the gene product
How to screen for the expression of the recombinant protein
Detect expression of protein on basis of size
Specific antibodies which recognise the protein
Detect the presence of expression tags which have been added to the protein during cloning process
Detect the biological activity: of the recombinant protein e.g. enzymic activity; of the expression tag
Expression tags
Added during the clone process to either end of the protein
Used to confirm that the gene product has been expressed- enzymic activity, immunogenicity
Can be used to aid purification of the expressed protein
Can be removed by enzymic cleavage
