BIO 2200 Exam 4 Flashcards
What is genetic engineering?
using in vitro techniques to alter genetic material in the laboratory
What do restriction enzymes do?
recognize specific DNA sequences and cut DNA at those sites; protect cell from invasion from foreign DNA
How many classes of restriction enzymes are there? What does type II do?
three; Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation
What do modification enzymes do?
Chemically modify nucleotides in restriction recognition sequence; protect cell’s DNA for restriction enzymes
What happens during gel electrophoresis? How does is it do that? What is the gel made of? What end does the DNA travel towards? What are the gels stained by and why?
It separates DNA molecules based on size. Electrophoresis uses an electrical field to separate charged molecules. Gels are usually made of agarose, a polysaccharide. Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups. Gels are stained with ethidium bromide so DNA can be visualized under UV light.
How can the size of DNA be determined?
Size of fragments can be determined by comparison to a standard
What is a restriction map?
a map of the location of restriction enzyme cuts on a segment of DNA
What is nucleic acid hybridization?
base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix
What is a nucleic acid probe?
segment of single-stranded DNA that is used in hybridization and has a predetermined identity
What is southern blot? northern blot?
Southern blot is a hybridization procedure where DNA is in the gel and probe is RNA or DNA. Northern blot is when RNA is in the gel.
What is molecular cloning?
isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
What are the three main steps of gene cloning?
1) Isolation and fragmentation of source DNA
2) Insertion of DNA fragment into cloning vector
3) Introduction of cloned DNA into host organism
What are most vectors derived from?
plasmids or viruses
What is DNA ligase?
enzyme that joins two DNA molecules (works with sticky or blunt ends)
What is a gene library? What process creates it?
Gene library is a mixture of cells containing a variety of genes. Shotgun cloning creates gene libraries by cloning random genome fragments.
What is site-directed mutagenesis? What can it do?
Site-directed mutagenesis is performed in vitro and introduces mutations at a precise location. Can be used to assess the activity of specific amino acids in a protein.
What is cassette mutagenesis?
When DNA fragment are cut, excised, and replaced by a synthetic DNA fragment (DNA cassettes or cartridges).
What is gene disruption? What can it cause?
Gene disruption is when cassettes are inserted into the middle of the gene. Gene disruption causes knockout mutations (gene becomes inoperative).
What are reporter genes? What are some examples?
Reporter genes encode proteins that are easy to detect and assay. Examples are lacZ, luciferase, GFP genes.
What are gene fusions?
When promoters or coding sequences of genes of interest are swapped with those of reporter genes to elucidate gene regulation under various conditions.
What are 4 reasons plasmids make good cloning vectors?
1) Small size –> easy to isolate DNA
2) Independent origin of replication
3) Multiple copy number: get multiple copies of cloned gene per cell
4) Presence of selectable markers
What is a common cloning vector? What does it contain?
pUC19 is a common cloning vector. It is a modified ColE1 plasmid that contains ampicillin resistance and lacZ genes. It also contains polylinker (multiple cloning site) within lacZ gene.
What is blue/white screening?
It is used to see what has foreign DNA inserted. Blue colonies do not have vector with foreign DNA inserted, while white colonies have foreign DNA inserted.
What is insertional inactivation? What does it cause?
It is when the lacZ gene is inactivated by insertion of foreign DNA. This inactivated lacZ then cannot process Xgal, which inhibits the blue color to develop.
What are 5 characteristics of ideal hosts for cloning vectors?
1) Capable of rapid growth in inexpensive medium
2) Nonpathogenic
3) Capable of incorporating DNA
4) Genetically stable in culture
5) Equipped with appropriate enzymes to allow replication of the vector
What are 3 examples of ideal hosts for cloning vectors?
Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae
What are 3 advantages and 2 disadvantages of E. coli being a host for cloning vector?
Advantages are that it is well-developed in genetics, has many strains available, and is the best known bacterium. Disadvantages are that it is potentially pathogenic and the periplasm traps proteins.
What are 4 advantages and 2 disadvantages of B. subtilis being a host for cloning vector?
Advantages are that it is easily transformed, nonpathogenic, naturally secretes proteins, and endospore formation simplifies culture. Disadvantages are that it is genetically unstable and genetics are less developed than in E. coli.
What are 4 advantages and 2 disadvantages of Saccharomyces cervisiae (eukaryote) being a host for cloning vector?
Advantages are that it is well-developed in genetics, nonpathogenic, can process mRNA and proteins, and easy to grow. Disadvantages are that plasmids are unstable and will not replicate most bacterial plasmids.
What is transfection? How is it done?
Transfection is introduction of DNA into mammalian cells. Originally done through phagocytosis of DNA by host cell. It can also be done using microinjection, electroporation, or gene gun.
What are shuttle vectors?
vectors that are stably maintained in two or more unrelated host organisms
What do expression vectors allow the experimenter to do?
allow experimenter to control the expression of cloned genes
What are 4 characteristics of expression vectors?
1) Based on transcriptional control
2) Allow for high levels of protein expression
3) Strong promoters (lac, trp, tac, trc, lambda PL)
4) Effective transcription terminators are used to prevent expression of other genes on the plasmid
What are 3 problems that can occur when mRNA produced must be efficiently translated?
1) Bacterial ribosome binding sites are not present in eukaryotic genomes
2) Differences in codon usage between organisms
3) Eukaryotic genes containing introns will not be expressed properly in prokaryotes
What are 5 examples of vectors that exist for cloning in eukaryotic cells?
Yeast artificial chromosomes (YACs), DNA virus SV40, adenovirus, vaccinia virus, and baculovirus
What do integrating vectors do?
Integrate into host chromosome and are stably maintained in cell
Why does the bacteriophage lambda make a good cloning vector?
1) Well-understood biology
2) Can hold larger amounts of DNA than most plasmids
3) DNA can be efficiently packaged in vitro
4) Can efficiently infect suitable host particles
What are the 5 steps to cloning with lambda?
1) Isolating vector DNA from phage particles and cutting it with the appropriate restriction enzyme
2) Connecting the lambda fragments to foreign DNA using DNA ligase
3) Packaging of the DNA by adding cell extracts containing the head and tail proteins
4) Infecting E. coli cells and isolating phage clones by picking plaques
5) Checking the recombinant phage for the presence of foreign DNA
What are cosmids? Why are they good cloning vectors (4)?
Cosmids are plasmid vectors containing the cos site from the lambda genome. They can be packaged into lambda virions, inserts as large as 50 kilo bases are accepted, avoids necessity of transforming E. coli, and phage particles are more stable than plasmids.
What are 3 specialized vectors for genome analysis?
Bacteriophage M13, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs)
What does the bacteriophage M13 do and what does it have?
clone DNA up to 5 kilo bases and contains lacZ for blue/white screening
What is the bacterial artificial chromosome constructed from and what is the host for it?
Constructed from the F plasmid and host for a BAC is generally a mutant strain of E. coli
What can yeast artificial chromosomes do and how to they replicate? What is similar to YACs?
YACs can accommodate 200-800 kilo bases of cloned DNA and they replicate like normal yeast chromosomes. Human artificial chromosomes are similar to YACs.
What is industrial microbiology? What did it originate from?
Uses microorganisms, typically grown on a large scale, to produce products or carry out chemical transformation. Originated with alcoholic fermentation processes.
What are 5 properties of a useful industrial microbe?
produces spores or can be easily inoculated, grows rapidly on a large scale in inexpensive medium, produces desired product quickly, should not be pathogenic, and amenable to genetic manipulation
What are 7 products of industrial microbiology?
microbial cells, enzymes, antibiotics, steroids, alkaloids, food additives, and commodity chemicals
What are commodity chemicals and what are some examples?
Inexpensive chemicals produced in bulk and include ethanol, citric acid, and many others
What is a primary metabolite? secondary metabolite?
Primary metabolites are produced during the exponential growth (alcohol), while secondary metabolites are produced during the stationary phase.
What are 5 characteristics of secondary metabolites?
They are not essential for growth, formation depends on growth conditions, produced as a group of related compounds, often significantly overproduced, and often produced by spore forming microbes during sporulation.
What do secondary metabolites require for production? Give an example. What do starting materials arise from?
Secondary metabolites are often large organic molecules that require a large number of specific enzymatic steps for production. For example, synthesis of tetracycline requires at least 72 separate enzymatic steps. Starting materials arise from major biosynthetic pathways.
What are fermentors?
A fermentor is where the microbiology process takes place. Any large-scale reaction is referred to as a fermentation (most are aerobic processes).
What must be done with industrial fermentors (4 things)?
They must be closely monitored during production run, growth and product formation must be measured, environmental factors must be controlled and altered as needed
(including temperature, pH, cell mass, nutrients, and product concentration), and
data on the process must be obtained in real time.
What is a scale-up? What are the steps?
The transfer of a process from a small laboratory scale to large-scale commercial equipment. Flask to laboratory fermentor to pilot plant to commercial fermentor.
What is the major task of a biochemical engineer?
they need to require the knowledge of the biology of producing organism and the physics of fermentor design and operation
What are antibiotics? What kind of metabolites are they? What are they produced by?
Antibiotics are compounds that kill or inhibit the growth of other microbes. They are typically secondary metabolites. Most antibiotics in clinical use are produced by filamentous fungi or actinomycetes.
How are antibiotics discovered?
Still discovered by laboratory screening. Microbes are obtained from nature in pure culture and then they are assayed for products that inhibit growth of test bacteria.
What is the cross-streak method used for?
The cross-streak method is used to test new microbial isolates for antibiotic production. Most isolates produce known antibiotics and most antibiotics fail toxicity and therapeutic tests in animals.
What is the time and cost of developing a new antibiotic?
Time and cost of developing a new antibiotic is approximately 15 years and $1 billion. It involves clinical trials and U.S. FDA approval.
What type of antibiotics are penicillins? What type of metabolite is penicillin production? Production only begins after what?
Penicillins are beta-lactam antibiotics. Penicillin production is typical of a secondary metabolite. Production only begins after near-exhaustion of carbon source. High levels of glucose repress penicillin production.
Production of what is second only to antibiotics in terms of total pharmaceutical sales?
vitamins; Vitamin B12 is produced exclusively by microorganisms. Riboflavin can also be produced by microbes.
What are amino acids used as (3) and what are 3 examples?
Amino acids are used as feed additives in the food industry, as nutritional supplements in nutraceutical industry, and as starting materials in the chemical industry. Examples are glutamic acid (MSG), aspartic acid and phenylalanine, and
lysine (food additives).
What are exoenzymes? What can they do?
Exoenzymes are enzymes that are excreted into the medium instead of being held within the cell. They are extracellular and they can digest insoluble polymers such as cellulose, protein, and starch.
Why are enzymes useful in industrial microbiology?
Enzymes are useful as industrial catalysts because they produce only one stereoisomer and have high substrate specificity.
What are enzymes produced from?
fungi and bacteria
What are bacterial proteases used in and what are they isolated from? Amylases and glucoamylases?
Bacterial proteases are used in laundry detergents (can also contain amylases, lipases, and reductases). They are isolated from alkaliphilic bacteria. Amylases and glucoamylases are commercially important because they produce high-fructose syrup.
What are extremozymes? What are they produced by?
Extremozymes are enzymes that function at some environmental extreme (pH or temperature). They are produced by extremophiles.
What are three ways to immobilize an enzyme? What are immobilized enzymes used in?
Three ways to immobilize an enzyme are bonding of enzyme to a carrier,
cross-linking of enzyme molecules, and enzyme inclusion. They are used in the starch processing industry.
What does wine come from? How big are the fermentors in which wine fermentation occurs and what are they made of? What is the difference between white wine and red wine?
Most wine is made from grapes. Wine fermentation occurs in fermentors ranging in size from 200 to 200,000 liters. Fermentors are made of oak, cement, glass-lined steel, or stone. White wine is made from white grapes or red grapes that have had their skin removed. Red wine is aged for months or years, while white wine is often sold without aging.
