chapter 8 Flashcards
what are the 4 STEPS- involved in genetic engineering to produce a protein product?
- recombinant DNA technology
- transformation
- cloning
- purification
describe what occurs during the recombinant DNA technology step
genetic code is identified and isolated from donor cell, pasted into a vector cell= recombinant DNA plasmid that carry the desired DNA code
describe what occurs during the transformation step
if genetically engineered cells express the new DNA, assays are conducted to test if the transformation occured by testing for the gene product
describe what occurs during the cloning step
transformed cells–> growing in culture
petri dishes–> broth cultures up to 10L–> up to 30,000L (manufacturing)
Majority of genetically engineered products are..
pharmaceuticals using recombinant DNA
examples of pharmaceutics genetically engineered products
neupogen, Cervarix
Probing DNA function
looking for a section on a gene that codes for protein of interest
define probe
a DNA or RNA molecules that is complementary to the DNA sequence
What do restriction enzymes do when probing DNA
chop up DNA into single strands
what does gel electrophoresis do when probing DNA
separate DNA
define the process of hybridization
probe molecules are “tagged” with either a radioactive
marker or a fluorescent label. The probes bounce around
the gel. If they bump into a complementary sequence,
they bind to it.
shows the location of the DNA sequence
define autoradiogram
record of hybridized complex
southern blotting define
blot, a gel with the
DNA fragments is transferred to a positively charged
membrane (the blot). The blot is then probed, then DNA is identified and removed for processing
What is the issue of mRNA probes?
degrades at high temperatures –> copy DNA (cDNA)
Describe how PCR is used for finding the gene of interest
use 2 primers (like probes) to recognize target DNA
PCR replicate primer, targeted gene and use a thermal cycler to generate millions of copies of DNA fragment s
define blot
membrane that has proteins
genetically engineered rennin molecule? why is it preferable to have genetically engineered rennin?
chymosin; expensive to harvest rennin
how is hybridization used in the lab
probe tech
two sets of enzymes used to cut & paste DNA
endonucleases- cut
DNA ligase- paste
difference between blunt and sticky ends
blunt ends- restriction enzymes that cut straight across DNA strand
sticky ends- one side longer than the other - allows for complementary matches
restriction fragment length polymorphisms analysis
studies of the differences in the fragment lengths that can read info about the difference in both sequences
DNA fingerprinting
RFLOP gives unique banding patterns on on a gel bc each person’s cod is unique
Where else is RFLP used?
in evolutionary studies that helps scientists understand DNA mutations that lead to differences in species
restriction enzymes mapping
determining the order of restriction sites of enzymes in relation to one another
transformation define
(human) recombinant plasmid DNA
transduction define
transformation viral DNA
Name of the process in which mammalian cells recieve and express rDNA
transfection
6 General Steps of a Transformation(bacteria uptake DNA)
- grow the host cells in broth culture
- keeping the cells on ice, make the competent with a treatment of calcium chloride or magnesium chloride
- Add the rDNA plasmids to the competent cells
- heat shock the cells from moving the cells from ice to a hot water bath = heat shock/cold shock
- add a nutrient broth for cells recovery & gene expression at optimum temperature
- place out the cells on some kind of selection media/ Agar that shows the cells are producing the new protein
What is the effect of adding cations to bacteria?
covers the membrane channel proteins in a positive charge- repel each other= expanded channels
easy for DNA molecules to move into the cell
Electroporation
cells made competent by exposing cells to an electric field
What is another way to allow cells to take in foreign DNA
heat and cold shock–> cells swell rapidly pulling DNA to membrane; when transferred back to cold it shrinks and tranps DNA inside = constant cycle improve transformations
Define recover period
this time, the cells are given nutrients in a sterile broth
and allowed to repair their membranes.
What is the selection process?
a method involving killing untransformed cells to see if they are properly producing new proteins from new genes
3 selection gene examples
antibiotic resistance gene
beta/galactosidase
green fluorescent protein gene
why do transformed cells need a recovery period
to recover from trauma and heal from weaken membrane
What is GFP and why is important in genetic
engineering work?
GFP- indicates the transformed cells with the glowing gene= indicates which cells are transformed
what is the scale up process
increases the number of transformed bacteria to a larger number
Why would the nutrient broth need to be increase during the scale up process?
as the volume of cells increase, so does the amount of product;
nutrient broth allows cells more room and more
nutrients.
what gets measured during scale-up assays
cell growth rate, product concentration, and product activity, and coontamination
recombinant α-amylase
an enzymatic activity assay may be used to measure the degree to which amylase’s
substrate, starch (or a synthetic substrate), is converted to a detectable product
(maltose).
What is another commonly used assay
ELISA
spinner flask
a sterile type of flask commonly used for scale up in which there is a spinner apparatus inside to keep cells suspended and aerated
define fermenters
automated containers used for fermentation or growth of microorganisms cultures designed to be easily monitored and controlled
ideal for larger cultures
What type of environmental conditions must be monitored
in cultures as they are scaled-up?
temperature, pH, nutrients, oxygen, protein concentration and activity
How is a QC department involved in the
manufacturing process?
check all characteristics important to FBLA
stability, potency and safety6
seed define
the initial colony or
a culture that is used as starter for a
larger volume of culture
exponential growth define
when a cell culture doubles in cell count with every cell cycle.
How quickly does cells grow and multiply in a culture?
20 minutes
Distinguish between the processes of alcoholic and lactic-
acid fermentation. Give an example of a product made by
each process.
Alcoholic fermentation converts pyruvate to ethanol and carbon dioxide
lactic acid fermentation converts pyruvate to lactic acid, both processes occurring in the absence of oxygen.
wine and beer (alcoholic fermentation)
yogurt and sauerkraut (lactic acid fermentation).
Place these events in order, from early in the product
pipeline to late in the pipeline.
Transformation Manufacturing
Fermentation R&D
Assay Development QC
Scale-up
R&D, Transformation, Assay Development, Fermentation, Scale-up, Manufacturing, QC
Which federal agency is responsible for setting
cGMP guidelines?
FDA
process of extracting plasmids from
cells
miniprep
a plasmid isolation that yields about 20 to 30 μg of DNA
midiprep
isolates plasmids from 15 to 25 mL of
culture with the goal of isolating several hundred micrograms of DNA.
maxiprep
starts with 100 mL or so of culture, giving
yields of over 500 μg of plasmid DNA.
Why would you do a prep?
to check if transformation has actually occur and to collect more plasmids for future transformations
3 methods to testing for the presence of DNA
- DNA indicator test
- Ethidium Bromide tests
- UV Spectrophotometer
How is plasmid DNA precipitated in the final steps of a
plasmid prep?
using alcohol & centrifugation
Once plasmid is extracted from a cell, how can a technician
know that it is the “correct” plasmid?
restriction enzymes & gel electrophoresis
define competent cells
treated to enhance their ability to take up and incorporate foreign DNA from their surrounding
define sodium dodecyl sulfate (SDS)
detergent that dissolves cell membranes
Define restriction enzymes mapping
cutting DNA into fragments & figuiring out their original position in a DNA molecules
5 basic steps of genetic engineering
find gene of interest
interest gene into vector
insert vector into host cells to produce more recombinant DNA
grow cells in culture to produce protein of interest
purify the proteins
miniprep 3 steps
explode the cells- to release contents of the cell
separate DNA by centrifugation, plasmids will be in the liquid supernatant
alcohol washes and further centrifugation clean & precipitate the plasmids into a pellet
3 methods to probe for gene of interest
fluorescent
gel electrophoresis or southern blotting
PCR
Name 4 possible errors during transformation
not adding cations or salts to create competent cells
not heating shocking at appropriate temperature
not adding nutrient broth in the recovery phase
incorrectly measuring plasmid DNA
Bacteria transformation flowchart
- Plasmid DNA is put with competent cells
- They are incubated and then put in a heating block
- Plasmid DNA enter competent cells through permealized membrane
- Recovery period
- Add growth medium
- Overnigh incubation at normal temperature
- put antibiotics in plate for incubation
define supernatant
the fluid above a pellet during centrifuge
Bacterial cell growth phases
lag
log- doubles
stationary
death 💀
what is the function of calcium chloride
increases transformation efficiency- cations & anions
