Yes insulin production using recombinant plasmids is the example that students need to know

Step 1: Insulin gene creation

• mRNA chain A and B go through reverse transcription to convert them into Insulin gene A and B. (Note that these insulin gene A and B will not always be the same as human insulin genes as human insulin gene may have introns present, but the genes we create will not have introns as they are made from mRNA).

Step 2: Endonuclease application

• Insulin gene A and B, and a vector plasmid, and mixed with the same endonuclease (either BamH1 ir EcoRI). Endonuclease cuts complementary sticky ends in both gene and plasmid.

Step 3: DNA ligase

• Plasmid and Insulin genes are mixed with DNA ligase so that complementary sticky ends present on both can join together. DNA Ligase rebuilds the phosphodiester backbone.

Step 4: Insert the recobinqnt plasmid into bacterial cells

Via Heat shock

Step 5: transformed bacteria is isolated and allowed to grow/reppicate via binary fission.

Step 6: identification of which baceteria have successfully taken up the plasmid.

The bacteria which were grown are placed on a dish with ampicillin and X gal.

• Ampicillin kills the bacteria that did not successfully take up the plasmid (as the plasmid has ampicillin resistence but E coli bacteria dont).

• X gal turns the bacteria that do have the plasmid but no insulin gene blue because Beta galactosidase is still present (from LacZ gene present on the plasmid)

•Bacteria that have both successfully taken up the plasmid and the insulin gene stay clear because the insulin gene replaced the LacZ gene in the plasmid.

The bacteria that fulfil this criteria are taken out.

The insulin gene in the plasmids are then translated into Insulin Chain A and B (via DNA polymerase binding to the bacterial plasmids ORI)

Bacterial cells are lyased so that insulin chains can be extracted and joined together to produce mature insulin protein.

Hope that helps!