Topic Isolating, Cloning and Sequencing DNA Essay - Main Points!

Q) Describe the mass production of human insulin using genetically modified E.coli cells 

Main points
Key idea:	Starting template for synthesis of complementary DNA (cDNA)
· Insulin mRNA extracted fr human pancreas, as template to syn complementary DNA cDNA by reverse transcriptase
Key idea:	Use of plasmid vector
· Plasmid vector containing two selection markers, antibiotic resistance gene (e.g. ampicillin resistance gene) + Lac Z gene
Key idea:	Formation of recombinant vector
· In separate rx, plasmid vector + cDNA digested w same restriction enz (e.g. SmaI), produce restriction frag w blunt ends · Terminal transferase - adds extra cytosine nucleotides to ends of cDNA and extra guanine nucleotides to the vector - to create complementary sticky ends b/w the two DNA mol · DNA ligase - form recombinant vector by joining cDNA to vector · By catalyzing phosphodiester bonds b/w the two DNA mol
Key idea:	Transformation of bacteria (e.g. E.coli) with recombinant vector + selection of transformed cells
· CaCl2 heat-shock method used · Antibiotics e.g. ampicillin (in Petri dish) selects for transformed bacteria w recombinant vector + those w re-annealed vector · Non-transformed cells die but transformed cells survive + divide - form bacterial colonies
Key idea:	Identifying the correct transformed colonies
· Ref to blue-white screening method · cDNA was previously inserted into Lac Z gene of vector, disrupts Lac Z gene seq · Incorrect / undesired transformed cells - took up re-annealed vectors (w intact Lac Z gene coding for β-galactosidase which turns X-gal substrate blue) · Result: blue colonies · Correctly transformed bacterial cells unable to produce functional β-galactosidase · Result: white colonies
Key idea:	Culture bacterial cells + isolation + purification of insulin
· After identifying correct (white) bacterial colonies, culture bacterial cells in a nutrient medium · Ref to expression of insulin gene to form protein facilitated by prokaryotic cell machinery · Ref to protein extraction + purification for use
Comments: (i) Visualize the answer with the aid of a labelled diagram. (ii) Verbalize the answer + write down the main points w/o referring to the answer (use abbreviations) (iii) Read again within the next 24h & once more within 72h (do not spend more than 10min)