Genetic Engineering Step 2 Study Guide
Explain the process used to perform a bacterial transformation. \n Restriction enzymes are used to isolate gene of interest.
Fragments are pasted into a plasmid using ligase.
Cells are made competent using calcium chloride (chemical) and heat/cold shocks (physical)
Cells are allowed to recover with nutrient broth
Cells are streaked on a plate and allowed to grow (selective media)
Assay of growth to check for production of product
QC & scale-up
Ā \n Vocabulary ā \n transformation- introducing foreign DNA into a bacteria through non-viral methods
transfection- introducing foreign DNA into a eukaryotic cell through non-viral methods
transduction- using a virus as a vector to to introduce genetic mater
endonucleases- (restriction enzymes) used to ācutā DNA at certain points/sequences \n DNA ligase- āglueā that pastes DNA together, used to make rDNA
competency- ability to take up DNA
heat/cold shocks- used to make cell competent, heat opens up pores to allow DNA to enter, cold shrinks pores to trap DNA inside
microinjection- use of a very small needle to inject DNA \n electroporation- use of electrical pulses to create pores that DNA can enter through
recovery period- time required for cells to repair their membrane after disruption
selection- use of selective media so that only transformed cells may grow
seed colony- large colony that is growing well, used to scale up
scale-up- growth of seed colony in large container
lag phase- cells adapt to environment
exponential phase- rapid growth
stationary phase- nutrients used up, cloudy broth \n death phase- all nutrients used up, bacteria die
fermentation- growing cells under optimum conditions for maximum cell division and product production
R&D- develop and conduct test and verify the products as they move through the product pipeline
QC- performs additional assays and more extensive testing \n preparation- extracting plasmid from transformed cells
DNA purity- based on absorption of light. 1.8 is the optimal value >2 is too high <1.5 is too low
transformation efficacy- number of transformed cells per plasmid DNA. \n (How effective transformation was)
ampicillin- antibiotic, used as a selection media to kill non-transformed cells \n arabinose- sugar to āturn onā GFP gene, start protein production
GFP gene- green fluorescent protein gene, found in jellyfish to make them glow in the presence of arabinose
nutrient broth- liquid growth media full of nutrients for bacteria
pGLO plasmid- DNA sequence that contains the gene for GFP and ampicillin resistance \n broth cultures- liquid growth cultures (nutrient broth is used for these)