Module 8 - Microbial Biotechnology Flashcards
What is biotechnology?
The exploitation of biological processes for industrial and other purposes
What are some common methods of biotechnology?
Genetic manipulation of microbes for production of proteins, antibiotics, hormones, and many other products
What has led to the development of molecular biology tools?
The understanding of genetic machinery of microbes
What can molecular biology tools be used for in biotechnology?
Used to develop microbes that can produce desired products
What is the intention of biotechnology?
To improve quality of human life
How has the biotechnology field changed over time?
It has grown very fast, and affecting almost all aspects of life
How come biotechnology has become widespread?
Due to the molecular biology tools developed
What are the three groups of biotechnology?
Red, white, and green
What is red biotechnology?
Medical applications
What is white biotechnology?
Industrial applications
What is green biotechnology?
Agricultural applications
What is bioprospecting?
Searching for useful new microbes to cultivate and add to collections
How does bioprospecting work?
Using a variety of strategies, scientists can isolate novel microbes from different environments, and test them for specific activities
What is metabolic/biochemical fermentation?
Catabolic reactants that produce ATP without oxygen
What is industrial fermentation?
The controlled and regulated aerobic/anaerobic culture of microbes to produce desired products
What is an example of biochemical fermentation?
Production of ethanol through yeast
What is the different between metabolic and industrial fermentation?
Metabolic fermentation has no oxygen, while industrial fermentation often uses oxygen
How is the majority of industrial fermentation carried out?
In the presence of oxygen
What is an example of industrial fermentation?
Large scale culture of E. coli for recombinant proteins
For the E. coli in industrial fermentation, what reaction is carried out?
Cellular respiration (in the presence of oxygen)
Where does industrial fermentation take place?
In bioreactors
What is another name for a bioreactor?
A fermenter
What do bioreactors do?
Control environmental conditions (nutrients, oxygen, pH, temperature) for industrial fermentation
What are the two basic types of bioreactors?
Fed-batch reactors and chemostats
How does a fed-batch reactor work?
High cell density is maintained by providing culture with growth limiting nutrients over time
Why does a fed-batch reactor provide growth limiting nutrients over time?
It prevents the production of non-desired side products
What is another name for a chemostat?
A continuous bioreactor
How does a chemostat work?
As new medium is added, the same amount of culture is removed
Why does a chemostats remove the same amount of culture as new medium is added?
It creates a precisely controlled constant growth rate of microbes
What does the choice of bioreactor depend on?
The type of microbe used and the nature of the desired end product
In practice, which bioreactor is more dependable and reproducible?
Fed-batch reactors
How do metabolites produced by cells depend on?
The growth phase of the microbes
What is the typical growth curve of a microorganism?
Lag phase, exponential phase, stationary phase, and death phase
To obtain the optimal production of a desired product, what might a researcher have to do?
Maintain the culture in a particular growth phase
How is the production of ethanol maximized in yeast?
They are maintained in the exponential phase with low oxygen and with glucose
For yeast growing under anoxic conditions, what is ethanol?
A primary metabolite
What is a primary metabolite?
The product of a metabolic process required for growth
What is a secondary metabolite?
A product not required for microbial growth
When is a secondary metabolite often produced?
During the stationary phase
What is an example of a secondary metabolite?
Antibiotics
When is a primary metabolite often produced?
During the exponential phase
What can genetic alterations in a microbe lead to (in biotechnology)?
Increased production of desired metabolites
What methods are used to mutate strains?
Random mutagenesis and site directed mutagenesis
How does random mutagenesis work?
Strains are exposed to mutagenic chemicals, and screened for a desired phenotype
What agents are used in random mutagenesis?
X-rays, UV light, or DNA damaging chemicals
What phenotypes would be screened for in random mutagenesis?
The production of an enzyme
What are the drawbacks of random mutagenesis?
The resulting mutations are undefined, they may be detrimental, and the screening can be difficult, labor-intensive, or costly
What is site directed mutagenesis?
A method to make specific mutations at specific known sites within a DNA molecule
What are the two types of site directed mutagenesis?
Oligonucleotide site-directed mutagenesis or PCR site-directed mutagenesis
What does oligonucleotide site-directed mutagenesis require?
Cloning of DNA in a vector that produces single stranded molecules
How does oligonucleotide site-directed mutagenesis work?
A complementary oligonucleotide with a desired sequence change is used for DNA synthesis in the presence of DNA polymerase
What happens after oligonucleotide site-directed mutagenesis?
Plasmids carrying the desired mutation can be recovered after transformation of E. coli
What does the oligonucleotide acts as in site-directed mutagenesis?
A primer for the DNA polymerase to continue on the plasmid
What does PCR site-directed mutagenesis require?
Complementary primers with the desired mutation
How does PCR site-directed mutagenesis work?
The complementary primers with the desired mutation will be used in a PCR reaction to create the mutation
What happens after PCR site-directed mutagenesis?
The PCR product is digested with Dpn1
What is Dpn1?
A restriction enzyme
What does Dpn1 do?
Cleaves the specific DNA site only when methylated during propagation of E. coli
What is the significance of Dpn1?
It will not cleave the DNA synthesized outside of the cell; it will only cleave the original DNA template replicated in the E. coli cell
What DNA does Dpn1 cleave?
The original DNA template in the E. coli cell (methylated)
What DNA does Dpn1 not cleave?
The mutated DNA template outside the E. coli cell (unmethylated)
What is the consequence of Dpn1 cleavage?
The desired product will be enriched
How can the presence of a correct mutation be verified?
By DNA sequencing
How much of the total cell protein in E. coli is the most abundant native protein?
~2%
What do expression vectors do?
Produce recombinant proteins to higher levels
How high can expression vectors drive the production of recombinant proteins?
~20%
How can recombinant proteins be produced?
Through expression vectors
What are some examples of recombinant proteins produced by expression vectors?
Insulin, human growth hormone, and antiviral interferons
What are the parts of an expression vector?
Customized promoters, an operator, an optimized ribosome binding site, a start and stop codon, transcriptional terminator sequences, a selectable marker gene, and an origin of replication
What is the significance of the promoters in an expression vector?
They drive high levels of transcription
What is the significance of the operator in an expression vector?
Regulate the level of transcription
What is the significance of the ribosome binding site in an expression vector?
It helps with proper translation
What is needed in a ribosome binding site for an expression vector?
A Shine-Dalgarno sequence
What is the significance of the start and stop codons in an expression vector?
Used in translation to make the polypeptide
What is the significance of the transcriptional terminator sequences in an expression vector?
To end transcription
What is the sequence of the start codon?
ATG
How are fusion proteins produced?
Through expression vectors
What are fusion proteins?
Proteins that contain two or more domains of other proteins
What is another name for fusion proteins?
Tagged proteins
What do expression vectors designed for expressing fusion proteins have?
A built in sequence for a peptide tag fused to the coding sequence of the protein of interest
What may tags be used for (in fusion proteins)?
For purification
What is an affinity tag?
A tag that facilitates purification
What are some examples of affinity tags?
Protein A, His tags, MBP, GST, and strep-tag
What does His tag stand for?
Histidine amino acids
Wat does MBP stand for?
Maltose-binding protein
What does GST stand for?
Glutathione S-transferase
What is the receptor for protein A?
Anitbodies
What is the affinity tag for antibodies?
Protein A
What is the receptor for His tags?
Ni2+
What is the affinity tag for Ni2+?
His tags
What is the receptor for MBP?
Maltose
What is the affinity tag for maltose?
MBP
What is the receptor for GST?
Glutathion
What is the affinity tag for glutathion?
GST
What is the receptor for Strep-tag?
Streptavidin
What is the affinity tag for streptavidin?
Strep-tag
What are the major uses of microbes in red biotechnology?
Production of secondary metabolites with therapeutic properties, and production of recombinant human proteins
True or false: antibiotics are the only secondary metabolite that have therapeutic effects
False: other metabolites have been found
True or false: all therapeutic agents are from microbes
False: some are from plants
What is an example of a therapeutic agent derived from plants?
The antimalarial compound artemisinin
What are antibiotics?
Chemicals that kill or inhibit growth of bacteria
Where are antibiotics produced in nature?
By soil bacteria and fungi
What are antibiotics commonly used for?
Treating bacterial infections
How come antibiotcs gives microbes an advanatge?
They kill off the competition for limited resources
What was the first antibiotic discovered?
Penicillin
What organism produces penicillin?
Penicillium fungi
What antibiotics were discovered in the early 1940s?
Streptomycin and actinomycin
Where was streptomycin and actinomycin found?
In culture filtrates of actinomyces and saprophytic filamentous soil bacteria
How are many of the commercially available antibiotics made?
Through Streptomyces bacteria
True or false: all secondary metabolites in red biotechnology are antimicrobial
False: some can help in biochemical reactions
What do statins do?
They inhibit cholesterol synthesis
What organism produces statins?
Fungi
How do statins work?
They block the active site of HMG-CoA reductase
What does HMG-CoA reductase do?
It is important in cholesterol synthesis
What are some fungi that produce statins?
Penicillium and Aspergillus
What has lowered the production costs for many drugs?
Improvements of strains and fermentation techniques
What is a type I interferon?
Interferons with antitumor and antiviral capacities
What are some examples of recombinant human proteins used in red biotechnology?
Type I interferons, factor 13A, and insulin
What is factor 13A, and what is it used for?
A blood coagulation factor that is used to treat hemophilia
Where is insulin naturally produced?
In the pancreas
What does insulin do?
Regulates blood sugar levels
How was insulin extracted before red biotechnology?
From pigs and cattle
Which is more cost-effective for insulin production: E. coli or yeast?
Yeast
How come yeast is more cost effective for insulin production?
No purification steps are needed
What needs to be done if insulin is made from E. coli?
The E. coli endotoxin must be removed
What are the major uses of microbes in white biotechnology?
To generate products in many sectors, such as chemicals, food, detergents, bioplastics, and biofuels
What is the goal of white biotechnology?
To reduce greenhouse gas emissions and move away from petrochemical based products
What is the basic principle of white biotechnology?
To use microbial conversion of low-cost biomass to products with higher value
What do oil refineries do?
Convert crude petroleum into a number of useable products
What is the problem with oil refineries?
They generate numerous pollutants
What is biorefinery?
The process of converting biomass feedstocks into a number of products (chemicals/energy) with fewer environmental concerns
What are some examples of biomass that is otherwise discarded?
Crop plants and forestry waste
What provides the starting material for fuel production?
Cellulose/hemicellulose
What can cellulose/hemicellulose lead to?
Fuel production (ethanol/butanol), biochemicals (succinic acid and acetic acid) and other biopolymers
What is cellulose?
A complex polymer of beta-1,4-linked glucose units
True or false: most microbes can break down cellulose
False: very few can break down cellulose
What needs to be done to cellulose before it can be used by microbes?
It needs to be degraded by physical processes or enzymes
What did original diesel engines run on?
Biodiesel produced from vegetable oil
How come there is a renewed interest for ethanol as a biofuel?
The environmental concerns with fossil fuels
How is the commercial production of ethanol carried out?
Specific strains of yeast (efficient production properties)
What is the maximum amount of alcohol an industrial yeast strain can tolerate?
~15%
How is alcohol concentration increased in white biotechnology?
Through distillation processes
What does the cost of alcohol production depend on?
The cost of biomass feedstock used
In North America, how is most of the ethanol produced?
From corn starch
What is done to the corn starch before being used for ethanol production?
It is broken down into glucose using amylase
What does amylase do?
Breaks down starch into glucose
True or false: engines would need major modifications to use ethanol
False: ethanol can be used in internal combustion engines with little modification
What is commercial ethanol commonly produced from?
Sugar and starch
True or false: ethanol can be produced from cellulose
False: this is still currently being developed
What is the key step in ethanol production?
The conversion of biomass into cellulose that can be fermented to produce alcohol
What are some (2) unintended consequences of ethanol fuel?
- An increased demand for sugar cane and corn starch may lead to increased prices for foods containing these products
- Large amounts of fertilizers are used, which can have long term effects
True or false: most waste biomass from agriculture can be used as feedstock
True: this can be a way to make the waste more useful
What waste biomass from agriculture can be used as feedstock?
Ligno-cellulose
What is ligno-cellulose?
A complex of cellulose, hemicellulose, and lignin
True or false: ligno-cellulose can be degraded by microbes
False: since it is a plant product, it cannot be broken down by microbes
What does cellulase do?
Converts ligno-cellulose into sugars
How is ligno-cellulose broken down?
Through cellulase
How can cellulase be used to make ethanol?
It can break ligno-cellulose into sugars, which can then be converted into alcohol by yeast
What is lignin?
A biodegradation-resistant phenolic polymer that surrounds polysaccharides
What is hemicellulose?
A complex polymer of hexoses, pentoses, and sugar acids
True or false: plastic waste is a major environmental problem
True: it can damage the environment
How can bioplastics serve as a solution to the plastic problem?
They are biodegradable, while other plastics are not
What do Bacillus megaterium bacteria produce?
PHB
What does PHB stand for?
Polyhydroxybutyrate
What bacteria produces PHB?
Bacillus megaterium
Besides PHB, what is another bioplastic polymer?
PHA
What does PHA stand for?
Polyhydroxyalkanoates
What bacteria produces PHA?
Multiple different types
How do bacteria use PHAs?
They are a carbon storage polymer for a source of energy in starvation
What can you say about materials constructed from PHAs?
They are biodegradable
Where have PHA synthesis genes been expressed?
In a number of plants
Why would PHA synthesis genes be expressed in plants?
These transgenic plants could then produce bioplastics
What is the current state of bioplastics research?
While the amount of production has been low, ongoing research is promising
What are the future goals of bioplastics?
Target PHA synthesis to specific parts of the plant (seed, leaf, stem), or produce PHA as a coproduct of crops (corn)
What does the production of many commercial products depend on?
The use of enzymes
What are some commercial products that depend on enzymes?
Food, detergents, and paper
Where do many of the enzymes for commercial products originate from?
Microbes
What is special about microbial enzymes?
They have high specificity and high efficiency, and are biodegradable
What does HFCS stand for?
High fructose corn syrup
Where is HFCS commonly found?
Soft drinks
How is HFCS made?
From amylase on corn starch
Where does the amylase enzyme for HFCS come from?
Bacillus bacteria
What is the advantage of HFCS?
It is much cheaper than sugar
What do several laundry detergents use?
A mixture of enzymes like lipases, amylases, proteases, glycosidases, and oxidases
What do the enzymes in laundry detergents do?
Work together to remove dirt and stains from fabrics
What is the largest single market for microbial enzymes?
The detergent industry
How do researchers try to find better enzymes for white biotechnology?
Through bioprospecting or improving existing enzymes to get better results
What is done to processed foods during production?
They are commonly fortified by adding compounds like vitamins
What is another name for vitamin B9?
Folic acid
Why is vitamin B9 added to foods?
It prevents growth defects in fetuses
What foods have added vitamin B9?
Breads and pastas
How are most vitamins synthesized?
Chemically
What vitamins are synthesized by microbes?
Vitamin B12 and Vitamin B2
What is another name for vitamin B12?
Cyanocobalamin
What is another name for vitamin B2?
Riboflavin
True or false: plants produce vitamin B12
False: they do not produce vitamin B12
How do animals get vitamin B12?
Through gut microbes
What can a vitamin B12 deficiency lead to?
Illness
True or false: vitamin B12 is produced chemically
False: it is complicated
What strains of bacteria are used to produce vitamin B12?
Pseudomonas or other bacterial strains
True or false: vitamin B2 is produced chemically
True: the synthesis is much simpler than vitamin B12
Which is simpler to produce chemically: vitamin B12 or vitamin B2?
Vitamin B2
Besides vitamins, what other food supplement is produced by microorganisms?
Amino acids
How are amino acids used in white biotechnology?
They are used in food, animal feed, nutritional supplement, and production of synthetic chemicals
What is L-glutamic acid used for?
A flavor enhancer
What is D/L-methionine used for?
Animal feed supplements
What is L-lysine used for?
Animal feed supplements
Which amino acid is used as a flavor enhancer?
L-glutamic acid
Which amino acid is used in animal feed supplements?
D/L-methionine and L-lysine
What are the most common amino acids produced in white biotechnology?
L-lysine, D/L-methionine, and L-glutamic acid
What is an advantage of microbial production of amino acids?
Stereospecificity
Which types of amino acids are produced from microbes?
L-isomers (which can be used by the human body)
Which types of amino acids does the human body use?
L-isomers (which can be produced by microbes)
What does present day agriculture involve?
The use of large amounts of insecticides, herbicides, and synthetic fertilizers
What is the problem with intensive agriculture?
It poses concerns for human health and the environment
What are the major uses of microbes in green biotechnology?
Pesticide/herbicide production, synthetic fertilizers, and introducing desired genes into plants
What is one of the most efficient ways to introduce DNA into plants?
Through transformation by Agrobacterium
What does Agrobacterium tumefaciens do?
Causes crown gull disease (tumors in many plants)
True or false: agrobacterium is needed for the initiation of the tumor
True: the bacterium was required for initiatino
True or false: agrobacterium is needed for the tumor to grow
False: the tumors continued to grow in the absence of the bacteria
How did Agrobacterium cause tumors in plants?
Through the pTi plasmid’s tDNA
How can the pTi plasmid move to other bacteria?
Through conjugation
How can genes be introduced into plants?
Through agrobacterium mediated plant transformation
What is agrobacterium mediated plant transformation?
Introducing new genes in a plant through tDNA mediated DNA transfer
What is an example of cross-kingdom transfer of DNA?
Agrobacterium mediated plant transformation
True or false: agrobacterium can be used to edit all plants
False: some plants cannot be edited this way
What plants cannot be edited through agrobacterium?
Wheat, barley, and rice
Besides agrobacterium, what are two methods to introduce DNA into a plant?
Protoplast formation and biolistics
What is protoplast formation?
The removal of the cell wall prior to DNA introduction
What is biolistics?
Using metal fragments coated with DNA, which are fired into the plant cells
What is Roundup?
A broad spectrum herbicide
Wat is the active ingredient in Roundup?
Glyphosate
True or false: plants can absorb glyphosate
True: plant tissues absorb glyphosate efficiently
What does glyphosate do?
It is a specific inhibitor of EPSP synthase
What does EPSP synthase do?
It is a key step in synthesizing aromatic amino acids
Which amino acids are aromatic?
Phenylalanine, tyrosine, and tryptophan
What is the effect of glyphosate on plants?
They cannot produce essential amino acids
True or false: glyphosate is toxic to humans and other mammals
False: it is most likely not toxic
How come glyphosate is not toxic to humans?
They don’t have the specific pathway (with EPSP synthase) to produce aromatic amino acids
How were glyphosate resistant transgenic plants generated?
By expressing an agrobacterium species CP4 EPSP synthase gene in several plants
What is special about CP4 EPSP synthase?
It is resistant to glyphosate
Which plants have had glyphosate tolerance incorporated?
Corn, cotton, and soybean
What is the advantage of having transgenic plants with CP4 EPSP synthase?
Farmers can use Roundup all year round
Which plasmid was used to introduce CP4 EPSP synthase into the plants?
PV-GMGT04
What does the PV-GMGT04 plasmid contain?
Two copies of EPSP synthase fused to CTP, plant virus promoters, and a selectable marker gene
What does CTP stand for?
Petunia Chloroplast Transit peptide
What is the selectable marker gene in PV-GMGT04?
uidA (beta-glucuronidase marker gene)
How was PV-GMGT04 introduced into the plants?
Through biolistics by Monsanto scientists
What significantly impacts the growth of plants?
Weeds and insects
What bacteria produce BT toxin?
Bacillus thuringiensis
What does Bacillus thuringiensis do?
It is a spore forming bacterium that produces intracellular protein crystals
What is another name for BT toxin?
Cry proteins
What does BT toxin do (generally)?
It has highly specific insecticidal activity against moths, butterflies, mosquitos, and beetle larvae
After being ingested, where is BT toxin activated?
In the insect gut
After activation, what does BT toxin do?
It binds to specific receptors on gut epithelial cells, producing pores in the cell membrane
What does BT toxin producing pores do?
It disrupts the osmotic balance, and kills the insect
Why would bacteria naturally produce BT toxin?
Killing these insects likely provided a source of nutrients for vegetative growth
What was the name of the original corn?
Teosinte
How did teosinte evolve into modern corn?
Farmers picked teosinte that produced larger ears of corn, grew better in local environments, or were resistant to diseases
At the very least, what must microbes be to be useful in biotechnology?
They must replicate well under standard laboratory conditions
How long does it take for microbes with superior qualities to be selected?
Years
How long does it take for crops with superior qualities to be selected?
Decades
What are culture collections?
Publicly available archives of isolated and characterized microbes
What is found in a culture collection?
Freeze-dried, frozen, or otherwise otherwise preserved living samples of microbial communities
What is one of the most important challenges in the scale-up from laboratory to industrial culture?
The transfer of oxygen and maintenance of adequate mixing at very large volumes
What is an example of a growth limiting nutrient in a Fed-batch reactor?
A carbon source
True or false: primary metabolites can be overproduced without affecting the culture
False: they often impeded growth
True or false: secondary metabolites can be overproduced without affecting the culture
True: they can produce toxicity or negative feedback
How come primary metabolites can affect the culture when overproduced?
They are intrinsically linked to energy-production pathways, which can inhibit growth
What genetic alterations can be done to make a microbe more useful for biotechnology?
- Manipulate expression of genes for enzyme production
- Downregulate competing pathways
- Grow on a lower-cost carbon source
What are some examples of DNA damaging chemicals?
Nitrosoguanidine and ethyl methanesulfonate
How was random mutagenesis used in red biotechnology?
It was used to produce strains of Penicillium chrysogenum with higher production of antibiotics
What Penicillium was first isolated by Alexander Fleming?
Penicillium notatum
What Penicillium was used for random mutagenesis for penicillin production?
Penicillium chrysogenum
What is genome editing?
A process of precisely modifying genomes directly
What was used for the first genome editing?
ZFNs and TALENs
What does ZFN stand for?
Zinc finger nucleases
What does TALEN stand for?
Transcription activator-like effector nucleases
What do ZFNs do?
They are well known motifs involved in recognizing and binding specific DNA sequences
What do TALENs do?
They are DNA binding proteins found in Xanthomonas bacteria
Where are TALENs found?
In Xanthomonas bacteria
How can ZFNs and TALENs be used in genome editing?
By changing the amino acids in the binding site, the proteins could be made to bind to almost any DNA sequence, and thus cleave at any specific DNA site
What is the difficulty of using ZFNs and TALENs in genome editing?
The design of the specificity of the DNA-binding domains is often tedious
What is the newest tool for genome editing?
CRISPR/Cas9
What does CRISPR/Cas9 do naturally?
Acts as a system to protect bacteria from bacteriophages
What is the difference between CRISPR/Cas9 and ZFNs/TALENs?
CRISPR/Cas9 does not rely on DNA-binding protein motifs
What does CRISPR stand for?
Clustered regulatory interspaced short palindromic repeats
What does Cas9 stand for?
CRISPR-associated enzyme
What does CRISPR/Cas9 do?
It can recognize DNA sequences by producing guide RNA molecules which can bind to complementary sequences
How can researchers modify the DNA sequence to be targeted by CRISPR/Cas9?
By changing the guide RNA sequence
How may improved functionality arise in a protein?
By adding more cysteine amino acids
How come adding more cysteine amino acids may help improve functionality of the protein?
Disulfide bonds stabilize proteins, making them less susceptible to denaturing conditions like heat or pH
What is an example of adding cysteine to stabilize a protein?
T4 lysozyme
What does T4 lysozyme do?
A bacterial cell wall degrading enzyme
Where was T4 lysozyme originally isolated from?
Phages
Where is T4 lysozyme used currently?
As a food preservative, and in infant formula
What does the successful alteration of a protein via site-directed mutagenesis require?
A fairly detailed knowledge of the three-dimensional structure of the protein
What is directed enzyme development?
Applying rounds of random mutagenesis and selection to achieve stepwise desired changes in a gene of interest
What does glycerol dehydrogenase do?
Catalyzes the formation of dihydroxyacetone
What does dihydroxyacetone do?
It is the main ingredient in sunless tanning products
What is error-prone PCR?
A method to produce variants of a particular enzyme
How does error-prone PCR work?
PCR is carried out under conditions with a very high error rate
How can the efficiency of directed enzyme development be increased?
Through DNA shuffling
What is the process for DNA shuffling?
Genes are digested and recombined in PCR to produce fusion, chimeric genes
How is DNA cleaved in DNA shuffling?
Through the enzyme DNase I
Why is DNA shuffling advanatgeous?
It can combine multiple beneficial variations into a single clone to create dramatically improved phenotypes
In microbial biotechnology, what is production limited by?
The genetic and physiological characteristics of the microbial strains used
How can the genetic and physiological characteristics of microbial strains be improved?
By making changes to the metabolic characteristics of the strain
What is an example of manipulating metabolic characteristics in microbial strains?
Genes can be introduced that allow the microbe to grow on a cheaper carbon source
How can high cell density of E. coli be achieved during fermentation?
To divert the precursor pyruvate away from the acetate pathway
How come the acetate pathway should be averted when growing E. coli for fermentation?
Lactate (a waste product) inhibits cell growth and protein production
How is the acetate pathway diverted in E. coli fermentation?
By using acetolactate synthase
What does acetolactase synthase do?
Converts pyruvate to acetoin, which is less inhibitory
What genes can be used to increase penicillin production?
cefEF and cefG
What does cefEF and cefG do?
They are involved in the biosynthesis of cephalosporin
What is needed (generally) for an expression vector to function?
The foreign (eukaryal) gene must have the correct components for bacterial transcription and translation
How can introns be removed from a gene?
By either artificial synthesis, or by using an mRNA transcript as the template for reverse transcriptase to make cDNA
What is the importance of the Shine-Dalgarno sequence?
Bacterial ribosomes recognize this sequence on the mRNA to initiate translation
What is glycosylation?
The addition of complex sugar molecules to polypeptides
What is the issue with glycosylation?
Many animal proteins have glycosylation, but bacteria are not capable of doing this
What is the purpose of glycosylation?
It is needed for proper folding of the protein
How are many mammalian proteins glycosylated?
With sialic acid
How are many bacterial and fungal proteins glycosylated?
With mannose
Which amino acids are commonly glycosylated?
Serine and threonine
What needs to be done if a eukaryal protein needs a specific glycosylation to function properly?
A eukaryal host (not a bacterial host) will be needed
Besides glycosylation, what may be a problem for bacterial hosts when making eukaryal proteins?
Disulfide bonds
What is used to produce recombinant proteins in insect hosts?
Modified insect baculoviruses
Which cell lines can be used for recombinant protein production?
Chinese hamster ovary cells, and HeLa cells
What is the disadvantage of eukaryal host lines for recombinant protein production?
They are more expensive and often do not produce as much recombinant protein
True or false: a Shine-Dalgarno sequence is needed in a eukaryal expression vector
False: eukaryal cells do not use the Shine-Dalgarno sequence
What is synthetic biology?
Constructing novel biological systems from constituent parts
How much can 1kb of dsDNA be synthesized for?
$250
What is the tricky part of synthetic biology?
Making the synthetic DNA functional
What must DNA do to be functional?
Undergo replication, transcription, and translation
What is needed to create a truly synthetic organism?
The entire microbial genome would need to be synthesized, introduced in a cell, replicate, and replace the preexisting host cell DNA
What does the synthetic organism E. coli do in xenobiology?
It can code for phosphoserine, a novel amino acid not found in nature, with the UAG stop codon
What is xenobiology?
The development of novel biological systems through expansion of the genetic code and incorporation of novel amino acids into proteins
How can xenobiology be useful?
It can enhance protein engineering and pathogen resistance in microorganisms
What needs to be done when considering the design of microbes for a specific function?
Genetic elements must be joined in different combinations and under appropriate regulation
What are some examples of biological parts?
Enzyme-encoding genes, regulatory DNA sequences, or genes encoding regulatory proteins
What is the goal with biological parts?
To produce these parts in a reproducible manner, so it can be combined with computer-aided design to create DNA that behaves in a predictable way
What does the concept of biological parts have its roots in?
Engineering disciplines
What is the issue with biology and standards?
Biology does not adhere to any standards, unlike other engineering parts
What is the purpose of the Registry of Standard Biological Parts?
To help give biological parts some standard, similar to other engineering disciplines
What does the Registry of Standard Biological Parts contain?
Descriptions of genetic parts (promoters, gene regulatory elements), ribosome binding sites and protein ORFs, and devices that are combinations of parts for protein production, reporter genes, and cell signaling
How is each part in the Registry of Standard Biological Parts stored?
In a BioBrick
What is a BioBrick?
A vector that is flanked by EcoRI and XbaI sites on one side, and SpeI and PstI sites on the other side
Why is XbaI and SpeI used in Biobricks?
They have the same core 4-nucleotide base sequence, only differing in flanking nucleotides. Thus, they have compatible sticky ends
What makes it possible for eventual designing of plasmids by BioBricks?
The lowering cost of DNA synthesis
What does HMG stand for?
Hydroxymethylglutaryl
Who discovered the first statin?
Akira Endo
What did Akira Endo do?
Discover the first statin
If insulin is decreased, what happens to the blood sugar levels?
They increase
How is insulin created in E. coli?
Two cultures have the two different chains (alpha and beta), which are fused together through disulfide bonds later
What is the advantage of using human insulin in bacteria over using pig or cattle insulin?
Human insulin is safer and more plentiful
What are some examples of fossil fuels?
Coal, oil, and natural gas
What are some examples of greenhouse gases?
CO2 (carbon dioxide) and N2O (nitrous oxide)
What products can be made from crude petroleum?
Gasoline, kerosene, wax, and asphalt
What does feedstock mean?
Raw starting material
What crops are specifically grown as biological feedstock?
Perennial grasses (Miscanthus), switchgrass, and poplar trees
How does distillation of alcohol work?
The culture is heated to evaporate the alcohol, which condenses around cooling coils. It can then be collected
In Brazil, what is the major source of ethanol?
Sugarcane
What problems are there with ethanol as a biofuel?
It only has 70% of the energy of gasoline, and it is more corrosive
What may be a better alternative to ethanol as a biofuel?
Butanol
What is butanol currently used for?
Latex, enamels, lacquers (make plastics flexible), and solvents
What are plastics?
Versatile polymers with a wide range of physical characteristics
What microbe was used for butanol fermentation?
Clostridium acetobutylicum
What is acetone used for?
Making cordite (the propellant used in military ammunition)
What are the two phases of butanol production?
- CO2 was bubbled to exclude O2 and produce acids (acetate, butyrate, hydrogen, and CO2)
- Acids were used to produce butanol, acetone, and ethanol
What was the typical yield of butanol after 60 hr?
12-20 g/L of solvent
How can PHB and PHA be altered for different properties?
By altering the length of the polymer, or the nature of the monomeric constituents
What is the key enzyme in PHA synthesis?
PHA synthase
What is the key enzyme in PHA degradation?
PHA depolymerase
What are the main goals of PHA bioplastics research?
- Lower the cost of production to make it competitive
2. Produce plastics with properties appropriate for specific uses
Which specialized devices can be made with PHA and still be cost-competative?
Medical devices such as sutures, vascular stents, and tissue engineering scaffolds
Why would researchers want plants to produce PHAs?
They can use the sun’s energy to make PHAs
What are some examples of enzymes used in white biotechnology?
Lipases, proteases, glycosidases, hydroxylases, nitrilases, acylases, and amidases
How is glucose isomerase used in soft drinks?
It adjusts the ration of glucose to fructose
What bacteria is glucose isomerase purified from?
Streptomyces sp.
Besides the laundry industry, what are some other key markets of enzymes in white biotechnology?
Baking, beverage, and dairy
How does a vitamin B12 deficiency occur?
Malabsorption in the gut (Crohn’s disease, inflammatory bowel syndrome)
Which fungi are used to synthesize vitamin B2?
Ashbya gossypii and Eremothecium ashbyii
What is aspartame composed of?
L-aspartic acid and L-phenylalanine
Which microbes are used to synthesize aspartame?
Bacillus flavum (L-aspartic acid) and Clostridium glutamicum (L-phenylalanine)
What are some features of microbial strains used for producing metabolites in white biotechnology?
They cannot survive and compete in their natural habitat because they are metabolically compromised
Which microbe is used to synthesize L-lysine?
Corynebacterium glutamicum
What is the key feedback enzyme in lysine production?
Aspartate kinase
What would the features of aspartate kinase be for a good microbe in biotechnology?
One where the regulatory allosteric site is non-functional, but where the active site is functional
How does lysine interact with aspartate kinase?
Through negative feedback
What is used to select a mutant capable of high production of a product?
An antimetabolite
What is an antimetabolite?
A compound that closely resembles the structure of a natural compound
What is the antimetabolite for lysine?
AEC
What dies AEC stand for?
S-2-aminoethylcysteine
How is AEC used to select for mutants that can produce lysine?
AEC and starting materials are added. If AEC binds to aspartate kinase, the cell will die. If it can’t, the cell will survive, and continue to produce lysine
What are some common traits introduced into plants in green biotechnology?
Taste, yield, nutritional content, pest and pathogen resistance, and shelf life
Which researchers first discovered agrobacterium transformation?
Erwin Smith and C. O. Townsend
How was conjugation mediated by agrobacterium?
Through opines
What are opines and what do they do?
Amino acid-like compounds that aid in conjugation in agrobacterium
What does tDNA stand for?
Transfer DNA
How does tDNA aid in tumor formation in plants?
The tDNA from pTi is integrated into the plant genome
How does tDNA cause tumors in plants?
It produces opines and plant hormones (auxins, cytokinins) that promote plant cell growth
What is the advantage of infecting plants with tDNA for agrobacterium?
They can feed on the opines produced through opine catabolism genes on pTi
How is tDNA transfered?
Through the vir genes on the pTi plasmid
What is another name for glyphosate?
N-phosphonomethyl-glycine
What is glyphosate derived from?
Glycine
What does EPSP stand for?
5-enolpyruvylshikimate-3-phosphate
What is the problem with glyphosate for heribicides?
It kills most crop plants too
What is the significance of CTP?
The plant EPSP synthase enzymes localize to the chloroplasts
Besides glyphosate, what are some other herbicides?
Glufosinate (inhibitor of glutamine synthetase enzyme) and 2,4-dichlorophenoxyacetic acid (2,4-D) (synthetic version of auxin phytohormone)
What are the problems with most insecticides?
They are potent neurotoxins that can have detrimental effects on humans, livestock, wildlife, and non-pest insects
What are the scientific names for the organisms that BT toxin is effective against?
Lepidopteran (moths and butterflies), dipteran (flies and mosquitos), and coleopteran (beetle) larvae
How does BT toxin become activated in the gut?
By dissolving in the alkaline conditions, and being digested by proteases
When was BT toxin first used as an insecticide?
1920s
In what bacterium are BT toxins expressed in now?
Pseudomonas fluorescens
