U3A1: 4E recombinant plasmids + bacterial transformation Flashcards
plasmid
small, circular loop of DNA seperate from the chromosome, typically found in bacteria.
recombinant plasmid
plasmid that has been edited to incorporate a target gene.
4 things needed to create a recombinant plasmid
- gene of interest
- plasmid vector
- restriction endonuclease
- DNA ligase
4 most important sites for a plasmid vector
- restriction endonuclease site
- antibiotic resistance genes
- origin of replication (ORI) (signals start site for DNA replication)
- reporter gene (identifiable phenotype)
bacterial transformation
process in which bacteria take up foreign DNA from the environment.
vector
the organism that is introduced to foreign DNA, typically plasmids.
role of gene of interest in creating a recombinant plasmid
gene of interest is the sequence of DNA that encodes the protein we wish to produce.
role of plasmid vector in creating a recombinant plasmid
piece of circular DNA that is modified to be an idea vector for bacterial transformation experiments.
role of restriction endonucleases in creating a recombinant plasmid
cuts the plasmid and gene of interest to create identical sticky ends on either end of the DNA sequence. the overhanging ends of the gene of interest will be complementary to the plasmid vector.
role of DNA ligase in creating a recombinant plasmid
joins the gene of interest to the plasmid vector by forming phosphodiester bonds in the sugar-phosphate backbone. creates the recombinant plasmid.
2 methods that promote recombinant plasmid uptake
- heat shock
- electroporation
process of heat shock
- bacteria and plasmids are placed in a (calcium ion) solution on ice.
- solution heated to 37-42 degrees for 25-45 seconds before being returned to ice.
- sudden change in temperature makes the plasma membrane more permeable and allows plasmid vectors to cross the phospholipid bilayer.
process of electroporation
- bacteria and plasmids get electrically shocked.
- the electric shock stops.
- electrical current causes plasma membrane to become more permeable and allows plasmid vectors to cross the phospholipid bilayer.
insulin
hormone responsible for regulating blood glucose levels.
making recombinant human insulin: process of creating the recombinant plasmid
- creating the recombinant plasmid.
- plasmid vectors prepared with ampR for antibiotic resistance and lacZ which acts as a reporter gene. (lacZ contains B-galactosidase enzyme which converts x-gal from colourless to blue)
- two plasmid vectors used: one for insulin A and one for insulin B. using a restriction endonuclease, both insulin samples are cut to form sticky ends. DNA ligase is used to re-establish the sugar-phosphate backbone and create 2 recombinant plasmids.
making recombinant human insulin: process of creating transformed bacteria
- creating transformed bacteria.
- plasmids are added to e. coli solution and either heat shock or electroporation can be used to increase uptake of plasmids.
- bacteria is spread and incubated on agar plates containing X-gal and antibiotic ampicillin. (colourless colonies can be determined to be transformed bacteria with the recombinant plasmid as their lacZ gene is dysfunctional since the gene of interest is located inside it.)
- recombinant plasmids will produce insulin subunit with a B-galactosidase tail formed from half of the transcribed+translated lacZ gene
making recombinant human insulin: process of protein production and extraction
- transformed bacteria that contain the recombinant plasmid are placed into conditions to exponentially reproduce before their membranes are broken down and the insulin produced is isolated and purified.
- two insulin chains have their B-galactosidase tails removed and are mixed together to form functional human insulin.
