Stages Of In Vivo Cloning Flashcards
In Vivo Cloning: Insertion Of DNA -> Vector
> Plasmid = cut open ( same restriction endonuclease )
> Creates same sticky ends as DNA fragment so the complementary bases are opposite eachother
> 2 strands of DNA = align
> DNA ligase joins them together ( forms sugar phosphate backbone )
In Vivo Cloning: Transformation
( Vector -> Host Cell )
> Host cell is where the gene will be expressed to create the desired protein
> The cell membrane needs to be more permeable to increase chance of vector entering the cytoplasm
> Host cells mixed with calcium ions & heat shocked
In Vivo Cloning: Identification: Antibiotic Resistance
> Plasmid with DNA fragment has resistance to ampicillin but not tetracycline
> Bacteria grown on agar, copy is transferred using a velvet block, stamped on top of agar plate & placed on top of new agar plate
> New agar plate has ampicillin dissolved in the agar, only bacteria which contains the plasmid can grow
> Same transfer again, but onto a plate which contains tetracycline in the agar
> The plasmid with the gene of interest has the tetracycline resistance gene interrupted, so the bacteria with the plasmid & fragment will not grow here
In Vivo Cloning: Identification: Fluorescence
> Jellyfish contain a gene which codes to create a green fluorescent protein ( GFP )
> This GFP gene is inserted into the plasmid
> DNA fragment is then inserted in the middle of the GFP gene - this disrupts it and prevents GFP production
> Only non flowing colonies contain the recombinant plasmid
In Vivo Cloning: Identification: Enzyme ( Lactase )
> Lactase can turn a certain colourless substance -> blue
> The gene for this enzyme is inserted into the plasmid
> DNA fragment inserted in the middle of this gene to disrupt it
> Bacteria is grown on agar with the colourless substance
> Those that stay colourless contain the plasmid and DNA fragment
In Vivo Cloning: Growth/Cloning
> Once it has been confirmed which bacteria have taken up the plasmid with the DNA fragment & gene of interest, large volumes are grown in a fermenter
> The recombinant DNA will produce a protein that can be extracted and purified to make the desired product that can be sold
