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What is the goal of genetic engineering?
What is the goal of genetic engineering?
The goal of genetic engineering is to produce organisms with new, improved characteristics.
What is the first step in isolating genetic information for gene engineering?
What is the first step in isolating genetic information for gene engineering?
What enzyme degrades bacterial cell walls?
What enzyme degrades bacterial cell walls?
Lysozyme.
What is the clear liquid left behind after a precipitate has been spun down to the bottom of a vessel by centrifugation?
What is the clear liquid left behind after a precipitate has been spun down to the bottom of a vessel by centrifugation?
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Probes used in DNA probing are always made from RNA molecules.
Probes used in DNA probing are always made from RNA molecules.
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What are primers used for in polymerase chain reaction (PCR)?
What are primers used for in polymerase chain reaction (PCR)?
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What is cDNA?
What is cDNA?
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What is the process called when cells take up and express foreign DNA called?
What is the process called when cells take up and express foreign DNA called?
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What type of DNA is often used as vectors to carry genes of interest into cells?
What type of DNA is often used as vectors to carry genes of interest into cells?
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What is the process called when viruses are used to transform cells?
What is the process called when viruses are used to transform cells?
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What are enzymes that cut RNA or DNA at specific sites called?
What are enzymes that cut RNA or DNA at specific sites called?
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What are the sticky ends in restriction fragments?
What are the sticky ends in restriction fragments?
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What enzyme is used to seal the pieces of DNA with matching sticky ends in the process of creating recombinant DNA?
What enzyme is used to seal the pieces of DNA with matching sticky ends in the process of creating recombinant DNA?
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What are the pieces of DNA that result from a restriction enzyme digestion called?
What are the pieces of DNA that result from a restriction enzyme digestion called?
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What are the differences in fragment lengths that result from restriction digestion due to differences in the genetic code for the same gene between two individuals called?
What are the differences in fragment lengths that result from restriction digestion due to differences in the genetic code for the same gene between two individuals called?
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What is the process of determining the order of restriction sites of enzymes in relation to each other called?
What is the process of determining the order of restriction sites of enzymes in relation to each other called?
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What is the ability of cells to take up DNA called?
What is the ability of cells to take up DNA called?
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What is the process of screening potential clones for the expression of a particular gene called?
What is the process of screening potential clones for the expression of a particular gene called?
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Heat and cold shocks can increase transformation efficiency by a thousand times.
Heat and cold shocks can increase transformation efficiency by a thousand times.
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What is the period following transformation where cells are given nutrients and allowed to repair their membranes and express the selection genes called?
What is the period following transformation where cells are given nutrients and allowed to repair their membranes and express the selection genes called?
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What is the process of increasing the size or volume of the production of a particular product called?
What is the process of increasing the size or volume of the production of a particular product called?
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What are the automated containers used for fermentation or growth of microorganism cultures designed to be easily monitored and controlled called?
What are the automated containers used for fermentation or growth of microorganism cultures designed to be easily monitored and controlled called?
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What is the initial colony or culture that is used as a starter for a larger volume of culture called?
What is the initial colony or culture that is used as a starter for a larger volume of culture called?
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What is the growth rate that bacteria maintain when they double in population size every cell cycle called?
What is the growth rate that bacteria maintain when they double in population size every cell cycle called?
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What is the initial period of growth for cells in culture after inoculation called?
What is the initial period of growth for cells in culture after inoculation called?
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What is the latter period of a culture in which growth is limited due to depletion of nutrients called?
What is the latter period of a culture in which growth is limited due to depletion of nutrients called?
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During the manufacture of a therapeutic product, what are the standards set and monitored by the FDA called?
During the manufacture of a therapeutic product, what are the standards set and monitored by the FDA called?
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What is the process of extracting plasmids from cells called?
What is the process of extracting plasmids from cells called?
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What type of DNA preparation yields approximately 20 ug/50uL of plasmid DNA?
What type of DNA preparation yields approximately 20 ug/50uL of plasmid DNA?
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What type of DNA preparation yields approximately 100-300ug/mL of plasmid DNA?
What type of DNA preparation yields approximately 100-300ug/mL of plasmid DNA?
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What type of DNA preparation yields approximately 500ug or more of plasmid DNA?
What type of DNA preparation yields approximately 500ug or more of plasmid DNA?
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A miniprep is used to confirm that a transformation has actually occurred as planned.
A miniprep is used to confirm that a transformation has actually occurred as planned.
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What is the common way to determine the concentration of DNA in a sample?
What is the common way to determine the concentration of DNA in a sample?
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What is the purity value of a sample?
What is the purity value of a sample?
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What is the preferred purity value for a plasmid preparation?
What is the preferred purity value for a plasmid preparation?
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If the purity value of a sample is over 2.0, the sample is probably contaminated with RNA.
If the purity value of a sample is over 2.0, the sample is probably contaminated with RNA.
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What can be used to reduce protein contamination in a DNA sample?
What can be used to reduce protein contamination in a DNA sample?
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After a DNA sample's purity and concentration are determined, what can be used to confirm that the plasmid purified is indeed the one desired?
After a DNA sample's purity and concentration are determined, what can be used to confirm that the plasmid purified is indeed the one desired?
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What treatment encourages cells to incorporate rDNA plasmids during a bacterial transformation?
What treatment encourages cells to incorporate rDNA plasmids during a bacterial transformation?
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What is a form of selection used to identify transformed cells?
What is a form of selection used to identify transformed cells?
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What is a spinner flask used for?
What is a spinner flask used for?
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Which of the following treatments will work to extract and isolate chromosomal DNA from bacterial cells?
Which of the following treatments will work to extract and isolate chromosomal DNA from bacterial cells?
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Which restriction enzyme will result in a blunt end cut?
Which restriction enzyme will result in a blunt end cut?
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Cutting DNA into fragments then figuring out their order is called what?
Cutting DNA into fragments then figuring out their order is called what?
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During scale up of cultured CHO cells cultures in a spinner flask, the cells grow for how long before being transferred to a larger culture?
During scale up of cultured CHO cells cultures in a spinner flask, the cells grow for how long before being transferred to a larger culture?
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After doing a plasmid preparation, the presence of DNA can be determined by what method?
After doing a plasmid preparation, the presence of DNA can be determined by what method?
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Give an example of a DNA vector
Give an example of a DNA vector
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What is the purpose of adding cations, calcium, or magnesium to host cells before a transformation?
What is the purpose of adding cations, calcium, or magnesium to host cells before a transformation?
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Why are plasmid preparations done?
Why are plasmid preparations done?
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How is DNA spliced?
How is DNA spliced?
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In a mini prep, how is DNA extracted in the final step?
In a mini prep, how is DNA extracted in the final step?
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When scaling up cell cultures, when are assays done?
When scaling up cell cultures, when are assays done?
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Before recombinant cells are reproduced, what must be done?
Before recombinant cells are reproduced, what must be done?
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What was rennin replaced with in cheese making and why?
What was rennin replaced with in cheese making and why?
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What is an example of product that could have been derived from genetic engineering?
What is an example of product that could have been derived from genetic engineering?
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What is the largest scale of producing transformed cells called?
What is the largest scale of producing transformed cells called?
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What happens during the stationary stage of cell growth?
What happens during the stationary stage of cell growth?
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A piece of DNA that results from being spliced into a vector is called what?
A piece of DNA that results from being spliced into a vector is called what?
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After the gene for the protein of interest has been identified, which method is used?
After the gene for the protein of interest has been identified, which method is used?
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What does it mean to probe DNA?
What does it mean to probe DNA?
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After cell lysis, the cell contents are removed from the solution leaving what?
After cell lysis, the cell contents are removed from the solution leaving what?
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On a UV spec, what wavelength does DNA absorb the maximum amount of light?
On a UV spec, what wavelength does DNA absorb the maximum amount of light?
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What is done during fermentation in terms of cell growth?
What is done during fermentation in terms of cell growth?
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What aspect of research is the host cell type considered for?
What aspect of research is the host cell type considered for?
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What does the FDA CG & P deal with?
What does the FDA CG & P deal with?
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Study Notes
Genetic Engineering Principles
- Genetic engineering manipulates genetic information to create organisms with enhanced traits.
- The process involves isolating a desired gene, placing it into a vector (rDNA), transferring it to a host cell, growing the recombinant cells, and purifying the resulting protein product.
Steps of Genetic Engineering
- Isolation: Isolate the target gene from the donor cell and ensure it is correctly identified. Insert into a suitable vector.
- Recombination: Introduce the vector containing the gene into a host cell using techniques like heat shock and ice bath. Cations (calcium, magnesium) can also increase transformation efficiency.
- Cloning/Production: Grow the host cells (clones) on a small, then large scale for product production, using various scale-up methods like spinner flasks.
- Purification (and Testing): Extract and purify the protein product followed by activity and quality control checks.
Isolating Gene Information
- Cell lysis (breaking open the cells) precedes DNA extraction and purification methods depend on the cell type.
- DNA is separated from cellular molecules, like RNA.
Bacterial Genomic DNA Extraction
- Requires a sufficient amount of bacterial culture (grown on agar or broth).
- The cells are treated with lysozyme (to break down the cell wall) in a buffered solution.
- Osmotic pressure causes cell rupture, releasing cell contents.
- Detergents dissolve membrane lipids, and proteins are precipitated (sometimes with help from proteases/salts).
- RNA removal is done through RNase addition.
- Centrifugation separates cell debris from DNA.
- DNA remains in the supernatant.
Bacterial Plasmid DNA Extraction
- Similar to bacterial chromosomal DNA extraction but with key differences.
- Alkaline lysis and neutralization are used for plasmid separation from cell debris and genomic DNA, which are precipitated out.
Probing for Genes
- Probes—complementary DNA/RNA sequences—are used to locate genes of interest.
- Restriction enzymes cut DNA into fragments for analysis, a process followed by gel electrophoresis.
- Southern blotting moves DNA fragments to membranes where probes can bind for visualization.
- cDNA (copy DNA), created from mRNA, is used to avoid mRNA degradation.
- Probes can be labelled with radioactive or fluorescent markers for detection. Visualization is done with autoradiograms or fluorescent microscopies.
Polymerase Chain Reaction (PCR)
- PCR uses primers to replicate specific DNA sequences (millions of copies) in hours.
- Primers are small DNA fragments that bind to target sequences.
Using Plasmids and Vectors
- Plasmids (small circular DNA) are used as vectors for transferring genes into cells.
- Cosmids are large plasmids used as vectors.
- Viruses are used as vectors for carrying genes into mammalian cells (called transduction) and vectors are cut with restriction enzymes and the desired genes are inserted.
Cell Transformation
- Transformation is the incorporation of foreign DNA into a cell and expression of that DNA.
- Heat shock and ice bath treatment encourages cells to incorporate rDNA plasmids. Antibiotic resistance genes are used to select the transformed cells.
- Techniques (like heat shock + CaCl₂) enhance uptake of plasmids by cells.
Scale-Up and Cell Culture Optimization
- Cultures are scaled up from small flasks (50 mL) to large fermenters (10,000 L or more).
- Conditions are carefully monitored to maximize product yield (nutrients, temperature, oxygen, and sterility). Spinner flasks are used during scale up to aerate cells.
- Assays that measure specific protein production are essential to track progress during scale-up.
- Quality Control (QC) and Assay Services monitor product quality and identify issues.
- Cell cultures grow for 3-4 days in spinner flasks before a larger culture.
Fermentation
- "Fermentation" refers to optimized growth conditions for maximum cell growth and product production in biotech.
- Various fermentation processes exist for different product synthesis (e.g., alcoholic, lactic acid).
- Growing cells from starter cultures (seeds) allows efficient exponential growth.
- Monitoring growth phases (lag phase, exponential growth) is crucial.
- Reaching the stationary phase requires transferring cells to new environments.
- Following current Good Manufacturing Practices (cGMP) is critical in the production process.
- Big tanks are used for growing transformed cells during fermentation.
Plasmid Preparation
- Minipreps (small), midipreps (medium), and maxipreps (large) yield different amounts of plasmid DNA for various purposes.
- Preparation involves extracting, purifying, and quantitating the DNA. Alcohol precipitation is used to extract DNA in the final step of a mini-prep.
DNA Quantification & Purity
- UV spectrophotometry is used to measure DNA concentration and purity.
- The 260/280 nm absorbance ratio determines the DNA's purity (at least 1.8 is good, closer to 2.0 is better).
- Contamination (like RNA or protein) can affect the ratio dramatically.
- Correct ratio will enable effective testing of DNA for transformation and restriction digestion. DNA is absorbed by light the most at 260 nm.
Restriction Enzymes and Mapping
- Restriction enzymes cut DNA at specific sequences.
- AluI creates blunt ends.
- Restriction enzyme mapping involves cutting DNA into fragments and determining their order.
Additional Information from Flashcards
- Bacterial Transformation: Heat shock and ice bath treatment increases plasmid uptake. Cations (calcium, magnesium) also enhance transformation efficiency.
- Transformed Cell Identification: Adding antibiotic resistant genes allows selection of transformed cells.
- Plasmid Preparation: Minipreps, midipreps, and maxipreps are used for different amounts of plasmid DNA for analysis or use. Cutting plasmids with restriction enzymes helps identify them.
- Scale Up: Assays are performed during scale-ups, between cultures and fermentation tanks.
- DNA Isolation & Confirmation: Confirming gene presence, isolation, and identification are critical before reproduction of cells.
- Genetically Equivalent Replacement: Rennin was replaced by the genetically engineered chymosin in cheese making.
- Probes: Used to locate specific genes of interest.
- Protein Production: Protein assays are used to determine the production levels of the protein of interest after gene identification.
- Host Cell Selection: Host cell type is considered during research and development (R&D) of methods for targeting genes to appropriate host cells and determining appropriate scale up and production processes.
- Manufacturing Scale: The largest scale of producing transformed cells is called manufacturing.
- Stationary Growth Phase: In the stationary stage of cell growth, cell growth and division slow down.
- rDNA: Recombinant DNA results from splicing a gene into a vector.
- DNA Splicing: Restriction enzymes are used to splice DNA.
- Restriction Mapping: A method cutting DNA into fragments then determining the order is restriction mapping.
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Description
This quiz explores the fundamental principles of genetic engineering, including gene isolation, vector introduction, cell cloning, and protein purification. It covers the entire process from the basic steps to the complex molecular techniques involved. Test your knowledge on how genetic traits are manipulated in organisms.
