Module 8 - Microbial Biotechnology Flashcards
1
Q
What is biotechnology?
A
The exploitation of biological processes for industrial and other purposes
2
Q
What are some common methods of biotechnology?
A
Genetic manipulation of microbes for production of proteins, antibiotics, hormones, and many other products
3
Q
What has led to the development of molecular biology tools?
A
The understanding of genetic machinery of microbes
4
Q
What can molecular biology tools be used for in biotechnology?
A
Used to develop microbes that can produce desired products
5
Q
What is the intention of biotechnology?
A
To improve quality of human life
6
Q
How has the biotechnology field changed over time?
A
It has grown very fast, and affecting almost all aspects of life
7
Q
How come biotechnology has become widespread?
A
Due to the molecular biology tools developed
8
Q
What are the three groups of biotechnology?
A
Red, white, and green
9
Q
What is red biotechnology?
A
Medical applications
10
Q
What is white biotechnology?
A
Industrial applications
11
Q
What is green biotechnology?
A
Agricultural applications
12
Q
What is bioprospecting?
A
Searching for useful new microbes to cultivate and add to collections
13
Q
How does bioprospecting work?
A
Using a variety of strategies, scientists can isolate novel microbes from different environments, and test them for specific activities
14
Q
What is metabolic/biochemical fermentation?
A
Catabolic reactants that produce ATP without oxygen
15
Q
What is industrial fermentation?
A
The controlled and regulated aerobic/anaerobic culture of microbes to produce desired products
16
Q
What is an example of biochemical fermentation?
A
Production of ethanol through yeast
17
Q
What is the different between metabolic and industrial fermentation?
A
Metabolic fermentation has no oxygen, while industrial fermentation often uses oxygen
18
Q
How is the majority of industrial fermentation carried out?
A
In the presence of oxygen
19
Q
What is an example of industrial fermentation?
A
Large scale culture of E. coli for recombinant proteins
20
Q
For the E. coli in industrial fermentation, what reaction is carried out?
A
Cellular respiration (in the presence of oxygen)
21
Q
Where does industrial fermentation take place?
A
In bioreactors
22
Q
What is another name for a bioreactor?
A
A fermenter
23
Q
What do bioreactors do?
A
Control environmental conditions (nutrients, oxygen, pH, temperature) for industrial fermentation
24
Q
What are the two basic types of bioreactors?
A
Fed-batch reactors and chemostats
25
How does a fed-batch reactor work?
High cell density is maintained by providing culture with growth limiting nutrients over time
26
Why does a fed-batch reactor provide growth limiting nutrients over time?
It prevents the production of non-desired side products
27
What is another name for a chemostat?
A continuous bioreactor
28
How does a chemostat work?
As new medium is added, the same amount of culture is removed
29
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
30
What does the choice of bioreactor depend on?
The type of microbe used and the nature of the desired end product
31
In practice, which bioreactor is more dependable and reproducible?
Fed-batch reactors
32
How do metabolites produced by cells depend on?
The growth phase of the microbes
33
What is the typical growth curve of a microorganism?
Lag phase, exponential phase, stationary phase, and death phase
34
To obtain the optimal production of a desired product, what might a researcher have to do?
Maintain the culture in a particular growth phase
35
How is the production of ethanol maximized in yeast?
They are maintained in the exponential phase with low oxygen and with glucose
36
For yeast growing under anoxic conditions, what is ethanol?
A primary metabolite
37
What is a primary metabolite?
The product of a metabolic process required for growth
38
What is a secondary metabolite?
A product not required for microbial growth
39
When is a secondary metabolite often produced?
During the stationary phase
40
What is an example of a secondary metabolite?
Antibiotics
41
When is a primary metabolite often produced?
During the exponential phase
42
What can genetic alterations in a microbe lead to (in biotechnology)?
Increased production of desired metabolites
43
What methods are used to mutate strains?
Random mutagenesis and site directed mutagenesis
44
How does random mutagenesis work?
Strains are exposed to mutagenic chemicals, and screened for a desired phenotype
45
What agents are used in random mutagenesis?
X-rays, UV light, or DNA damaging chemicals
46
What phenotypes would be screened for in random mutagenesis?
The production of an enzyme
47
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
48
What is site directed mutagenesis?
A method to make specific mutations at specific known sites within a DNA molecule
49
What are the two types of site directed mutagenesis?
Oligonucleotide site-directed mutagenesis or PCR site-directed mutagenesis
50
What does oligonucleotide site-directed mutagenesis require?
Cloning of DNA in a vector that produces single stranded molecules
51
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
52
What happens after oligonucleotide site-directed mutagenesis?
Plasmids carrying the desired mutation can be recovered after transformation of E. coli
53
What does the oligonucleotide acts as in site-directed mutagenesis?
A primer for the DNA polymerase to continue on the plasmid
54
What does PCR site-directed mutagenesis require?
Complementary primers with the desired mutation
55
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
56
What happens after PCR site-directed mutagenesis?
The PCR product is digested with Dpn1
57
What is Dpn1?
A restriction enzyme
58
What does Dpn1 do?
Cleaves the specific DNA site only when methylated during propagation of E. coli
59
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
60
What DNA does Dpn1 cleave?
The original DNA template in the E. coli cell (methylated)
61
What DNA does Dpn1 not cleave?
The mutated DNA template outside the E. coli cell (unmethylated)
62
What is the consequence of Dpn1 cleavage?
The desired product will be enriched
63
How can the presence of a correct mutation be verified?
By DNA sequencing
64
How much of the total cell protein in E. coli is the most abundant native protein?
~2%
65
What do expression vectors do?
Produce recombinant proteins to higher levels
66
How high can expression vectors drive the production of recombinant proteins?
~20%
67
How can recombinant proteins be produced?
Through expression vectors
68
What are some examples of recombinant proteins produced by expression vectors?
Insulin, human growth hormone, and antiviral interferons
69
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
70
What is the significance of the promoters in an expression vector?
They drive high levels of transcription
71
What is the significance of the operator in an expression vector?
Regulate the level of transcription
72
What is the significance of the ribosome binding site in an expression vector?
It helps with proper translation
73
What is needed in a ribosome binding site for an expression vector?
A Shine-Dalgarno sequence
74
What is the significance of the start and stop codons in an expression vector?
Used in translation to make the polypeptide
75
What is the significance of the transcriptional terminator sequences in an expression vector?
To end transcription
76
What is the sequence of the start codon?
ATG
77
How are fusion proteins produced?
Through expression vectors
78
What are fusion proteins?
Proteins that contain two or more domains of other proteins
79
What is another name for fusion proteins?
Tagged proteins
80
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
81
What may tags be used for (in fusion proteins)?
For purification
82
What is an affinity tag?
A tag that facilitates purification
83
What are some examples of affinity tags?
Protein A, His tags, MBP, GST, and strep-tag
84
What does His tag stand for?
Histidine amino acids
85
Wat does MBP stand for?
Maltose-binding protein
86
What does GST stand for?
Glutathione S-transferase
87
What is the receptor for protein A?
Anitbodies
88
What is the affinity tag for antibodies?
Protein A
89
What is the receptor for His tags?
Ni2+
90
What is the affinity tag for Ni2+?
His tags
91
What is the receptor for MBP?
Maltose
92
What is the affinity tag for maltose?
MBP
93
What is the receptor for GST?
Glutathion
94
What is the affinity tag for glutathion?
GST
95
What is the receptor for Strep-tag?
Streptavidin
96
What is the affinity tag for streptavidin?
Strep-tag
97
What are the major uses of microbes in red biotechnology?
Production of secondary metabolites with therapeutic properties, and production of recombinant human proteins
98
True or false: antibiotics are the only secondary metabolite that have therapeutic effects
False: other metabolites have been found
99
True or false: all therapeutic agents are from microbes
False: some are from plants
100
What is an example of a therapeutic agent derived from plants?
The antimalarial compound artemisinin
101
What are antibiotics?
Chemicals that kill or inhibit growth of bacteria
102
Where are antibiotics produced in nature?
By soil bacteria and fungi
103
What are antibiotics commonly used for?
Treating bacterial infections
104
How come antibiotcs gives microbes an advanatge?
They kill off the competition for limited resources
105
What was the first antibiotic discovered?
Penicillin
106
What organism produces penicillin?
Penicillium fungi
107
What antibiotics were discovered in the early 1940s?
Streptomycin and actinomycin
108
Where was streptomycin and actinomycin found?
In culture filtrates of actinomyces and saprophytic filamentous soil bacteria
109
How are many of the commercially available antibiotics made?
Through Streptomyces bacteria
110
True or false: all secondary metabolites in red biotechnology are antimicrobial
False: some can help in biochemical reactions
111
What do statins do?
They inhibit cholesterol synthesis
112
What organism produces statins?
Fungi
113
How do statins work?
They block the active site of HMG-CoA reductase
114
What does HMG-CoA reductase do?
It is important in cholesterol synthesis
115
What are some fungi that produce statins?
Penicillium and Aspergillus
116
What has lowered the production costs for many drugs?
Improvements of strains and fermentation techniques
117
What is a type I interferon?
Interferons with antitumor and antiviral capacities
118
What are some examples of recombinant human proteins used in red biotechnology?
Type I interferons, factor 13A, and insulin
119
What is factor 13A, and what is it used for?
A blood coagulation factor that is used to treat hemophilia
120
Where is insulin naturally produced?
In the pancreas
121
What does insulin do?
Regulates blood sugar levels
122
How was insulin extracted before red biotechnology?
From pigs and cattle
123
Which is more cost-effective for insulin production: E. coli or yeast?
Yeast
124
How come yeast is more cost effective for insulin production?
No purification steps are needed
125
What needs to be done if insulin is made from E. coli?
The E. coli endotoxin must be removed
126
What are the major uses of microbes in white biotechnology?
To generate products in many sectors, such as chemicals, food, detergents, bioplastics, and biofuels
127
What is the goal of white biotechnology?
To reduce greenhouse gas emissions and move away from petrochemical based products
128
What is the basic principle of white biotechnology?
To use microbial conversion of low-cost biomass to products with higher value
129
What do oil refineries do?
Convert crude petroleum into a number of useable products
130
What is the problem with oil refineries?
They generate numerous pollutants
131
What is biorefinery?
The process of converting biomass feedstocks into a number of products (chemicals/energy) with fewer environmental concerns
132
What are some examples of biomass that is otherwise discarded?
Crop plants and forestry waste
133
What provides the starting material for fuel production?
Cellulose/hemicellulose
134
What can cellulose/hemicellulose lead to?
Fuel production (ethanol/butanol), biochemicals (succinic acid and acetic acid) and other biopolymers
135
What is cellulose?
A complex polymer of beta-1,4-linked glucose units
136
True or false: most microbes can break down cellulose
False: very few can break down cellulose
137
What needs to be done to cellulose before it can be used by microbes?
It needs to be degraded by physical processes or enzymes
138
What did original diesel engines run on?
Biodiesel produced from vegetable oil
139
How come there is a renewed interest for ethanol as a biofuel?
The environmental concerns with fossil fuels
140
How is the commercial production of ethanol carried out?
Specific strains of yeast (efficient production properties)
141
What is the maximum amount of alcohol an industrial yeast strain can tolerate?
~15%
142
How is alcohol concentration increased in white biotechnology?
Through distillation processes
143
What does the cost of alcohol production depend on?
The cost of biomass feedstock used
144
In North America, how is most of the ethanol produced?
From corn starch
145
What is done to the corn starch before being used for ethanol production?
It is broken down into glucose using amylase
146
What does amylase do?
Breaks down starch into glucose
147
True or false: engines would need major modifications to use ethanol
False: ethanol can be used in internal combustion engines with little modification
148
What is commercial ethanol commonly produced from?
Sugar and starch
149
True or false: ethanol can be produced from cellulose
False: this is still currently being developed
150
What is the key step in ethanol production?
The conversion of biomass into cellulose that can be fermented to produce alcohol
151
What are some (2) unintended consequences of ethanol fuel?
1. An increased demand for sugar cane and corn starch may lead to increased prices for foods containing these products
2. Large amounts of fertilizers are used, which can have long term effects
152
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
153
What waste biomass from agriculture can be used as feedstock?
Ligno-cellulose
154
What is ligno-cellulose?
A complex of cellulose, hemicellulose, and lignin
155
True or false: ligno-cellulose can be degraded by microbes
False: since it is a plant product, it cannot be broken down by microbes
156
What does cellulase do?
Converts ligno-cellulose into sugars
157
How is ligno-cellulose broken down?
Through cellulase
158
How can cellulase be used to make ethanol?
It can break ligno-cellulose into sugars, which can then be converted into alcohol by yeast
159
What is lignin?
A biodegradation-resistant phenolic polymer that surrounds polysaccharides
160
What is hemicellulose?
A complex polymer of hexoses, pentoses, and sugar acids
161
True or false: plastic waste is a major environmental problem
True: it can damage the environment
162
How can bioplastics serve as a solution to the plastic problem?
They are biodegradable, while other plastics are not
163
What do Bacillus megaterium bacteria produce?
PHB
164
What does PHB stand for?
Polyhydroxybutyrate
165
What bacteria produces PHB?
Bacillus megaterium
166
Besides PHB, what is another bioplastic polymer?
PHA
167
What does PHA stand for?
Polyhydroxyalkanoates
168
What bacteria produces PHA?
Multiple different types
169
How do bacteria use PHAs?
They are a carbon storage polymer for a source of energy in starvation
170
What can you say about materials constructed from PHAs?
They are biodegradable
171
Where have PHA synthesis genes been expressed?
In a number of plants
172
Why would PHA synthesis genes be expressed in plants?
These transgenic plants could then produce bioplastics
173
What is the current state of bioplastics research?
While the amount of production has been low, ongoing research is promising
174
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)
175
What does the production of many commercial products depend on?
The use of enzymes
176
What are some commercial products that depend on enzymes?
Food, detergents, and paper
177
Where do many of the enzymes for commercial products originate from?
Microbes
178
What is special about microbial enzymes?
They have high specificity and high efficiency, and are biodegradable
179
What does HFCS stand for?
High fructose corn syrup
180
Where is HFCS commonly found?
Soft drinks
181
How is HFCS made?
From amylase on corn starch
182
Where does the amylase enzyme for HFCS come from?
Bacillus bacteria
183
What is the advantage of HFCS?
It is much cheaper than sugar
184
What do several laundry detergents use?
A mixture of enzymes like lipases, amylases, proteases, glycosidases, and oxidases
185
What do the enzymes in laundry detergents do?
Work together to remove dirt and stains from fabrics
186
What is the largest single market for microbial enzymes?
The detergent industry
187
How do researchers try to find better enzymes for white biotechnology?
Through bioprospecting or improving existing enzymes to get better results
188
What is done to processed foods during production?
They are commonly fortified by adding compounds like vitamins
189
What is another name for vitamin B9?
Folic acid
190
Why is vitamin B9 added to foods?
It prevents growth defects in fetuses
191
What foods have added vitamin B9?
Breads and pastas
192
How are most vitamins synthesized?
Chemically
193
What vitamins are synthesized by microbes?
Vitamin B12 and Vitamin B2
194
What is another name for vitamin B12?
Cyanocobalamin
195
What is another name for vitamin B2?
Riboflavin
196
True or false: plants produce vitamin B12
False: they do not produce vitamin B12
197
How do animals get vitamin B12?
Through gut microbes
198
What can a vitamin B12 deficiency lead to?
Illness
199
True or false: vitamin B12 is produced chemically
False: it is complicated
200
What strains of bacteria are used to produce vitamin B12?
Pseudomonas or other bacterial strains
201
True or false: vitamin B2 is produced chemically
True: the synthesis is much simpler than vitamin B12
202
Which is simpler to produce chemically: vitamin B12 or vitamin B2?
Vitamin B2
203
Besides vitamins, what other food supplement is produced by microorganisms?
Amino acids
204
How are amino acids used in white biotechnology?
They are used in food, animal feed, nutritional supplement, and production of synthetic chemicals
205
What is L-glutamic acid used for?
A flavor enhancer
206
What is D/L-methionine used for?
Animal feed supplements
207
What is L-lysine used for?
Animal feed supplements
208
Which amino acid is used as a flavor enhancer?
L-glutamic acid
209
Which amino acid is used in animal feed supplements?
D/L-methionine and L-lysine
210
What are the most common amino acids produced in white biotechnology?
L-lysine, D/L-methionine, and L-glutamic acid
211
What is an advantage of microbial production of amino acids?
Stereospecificity
212
Which types of amino acids are produced from microbes?
L-isomers (which can be used by the human body)
213
Which types of amino acids does the human body use?
L-isomers (which can be produced by microbes)
214
What does present day agriculture involve?
The use of large amounts of insecticides, herbicides, and synthetic fertilizers
215
What is the problem with intensive agriculture?
It poses concerns for human health and the environment
216
What are the major uses of microbes in green biotechnology?
Pesticide/herbicide production, synthetic fertilizers, and introducing desired genes into plants
217
What is one of the most efficient ways to introduce DNA into plants?
Through transformation by Agrobacterium
218
What does Agrobacterium tumefaciens do?
Causes crown gull disease (tumors in many plants)
219
True or false: agrobacterium is needed for the initiation of the tumor
True: the bacterium was required for initiatino
220
True or false: agrobacterium is needed for the tumor to grow
False: the tumors continued to grow in the absence of the bacteria
221
How did Agrobacterium cause tumors in plants?
Through the pTi plasmid's tDNA
222
How can the pTi plasmid move to other bacteria?
Through conjugation
223
How can genes be introduced into plants?
Through agrobacterium mediated plant transformation
224
What is agrobacterium mediated plant transformation?
Introducing new genes in a plant through tDNA mediated DNA transfer
225
What is an example of cross-kingdom transfer of DNA?
Agrobacterium mediated plant transformation
226
True or false: agrobacterium can be used to edit all plants
False: some plants cannot be edited this way
227
What plants cannot be edited through agrobacterium?
Wheat, barley, and rice
228
Besides agrobacterium, what are two methods to introduce DNA into a plant?
Protoplast formation and biolistics
229
What is protoplast formation?
The removal of the cell wall prior to DNA introduction
230
What is biolistics?
Using metal fragments coated with DNA, which are fired into the plant cells
231
What is Roundup?
A broad spectrum herbicide
232
Wat is the active ingredient in Roundup?
Glyphosate
233
True or false: plants can absorb glyphosate
True: plant tissues absorb glyphosate efficiently
234
What does glyphosate do?
It is a specific inhibitor of EPSP synthase
235
What does EPSP synthase do?
It is a key step in synthesizing aromatic amino acids
236
Which amino acids are aromatic?
Phenylalanine, tyrosine, and tryptophan
237
What is the effect of glyphosate on plants?
They cannot produce essential amino acids
238
True or false: glyphosate is toxic to humans and other mammals
False: it is most likely not toxic
239
How come glyphosate is not toxic to humans?
They don't have the specific pathway (with EPSP synthase) to produce aromatic amino acids
240
How were glyphosate resistant transgenic plants generated?
By expressing an agrobacterium species CP4 EPSP synthase gene in several plants
241
What is special about CP4 EPSP synthase?
It is resistant to glyphosate
242
Which plants have had glyphosate tolerance incorporated?
Corn, cotton, and soybean
243
What is the advantage of having transgenic plants with CP4 EPSP synthase?
Farmers can use Roundup all year round
244
Which plasmid was used to introduce CP4 EPSP synthase into the plants?
PV-GMGT04
245
What does the PV-GMGT04 plasmid contain?
Two copies of EPSP synthase fused to CTP, plant virus promoters, and a selectable marker gene
246
What does CTP stand for?
Petunia Chloroplast Transit peptide
247
What is the selectable marker gene in PV-GMGT04?
uidA (beta-glucuronidase marker gene)
248
How was PV-GMGT04 introduced into the plants?
Through biolistics by Monsanto scientists
249
What significantly impacts the growth of plants?
Weeds and insects
250
What bacteria produce BT toxin?
Bacillus thuringiensis
251
What does Bacillus thuringiensis do?
It is a spore forming bacterium that produces intracellular protein crystals
252
What is another name for BT toxin?
Cry proteins
253
What does BT toxin do (generally)?
It has highly specific insecticidal activity against moths, butterflies, mosquitos, and beetle larvae
254
After being ingested, where is BT toxin activated?
In the insect gut
255
After activation, what does BT toxin do?
It binds to specific receptors on gut epithelial cells, producing pores in the cell membrane
256
What does BT toxin producing pores do?
It disrupts the osmotic balance, and kills the insect
257
Why would bacteria naturally produce BT toxin?
Killing these insects likely provided a source of nutrients for vegetative growth
258
What was the name of the original corn?
Teosinte
259
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
260
At the very least, what must microbes be to be useful in biotechnology?
They must replicate well under standard laboratory conditions
261
How long does it take for microbes with superior qualities to be selected?
Years
262
How long does it take for crops with superior qualities to be selected?
Decades
263
What are culture collections?
Publicly available archives of isolated and characterized microbes
264
What is found in a culture collection?
Freeze-dried, frozen, or otherwise otherwise preserved living samples of microbial communities
265
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
266
What is an example of a growth limiting nutrient in a Fed-batch reactor?
A carbon source
267
True or false: primary metabolites can be overproduced without affecting the culture
False: they often impeded growth
268
True or false: secondary metabolites can be overproduced without affecting the culture
True: they can produce toxicity or negative feedback
269
How come primary metabolites can affect the culture when overproduced?
They are intrinsically linked to energy-production pathways, which can inhibit growth
270
What genetic alterations can be done to make a microbe more useful for biotechnology?
1. Manipulate expression of genes for enzyme production
2. Downregulate competing pathways
3. Grow on a lower-cost carbon source
271
What are some examples of DNA damaging chemicals?
Nitrosoguanidine and ethyl methanesulfonate
272
How was random mutagenesis used in red biotechnology?
It was used to produce strains of Penicillium chrysogenum with higher production of antibiotics
273
What Penicillium was first isolated by Alexander Fleming?
Penicillium notatum
274
What Penicillium was used for random mutagenesis for penicillin production?
Penicillium chrysogenum
275
What is genome editing?
A process of precisely modifying genomes directly
276
What was used for the first genome editing?
ZFNs and TALENs
277
What does ZFN stand for?
Zinc finger nucleases
278
What does TALEN stand for?
Transcription activator-like effector nucleases
279
What do ZFNs do?
They are well known motifs involved in recognizing and binding specific DNA sequences
280
What do TALENs do?
They are DNA binding proteins found in Xanthomonas bacteria
281
Where are TALENs found?
In Xanthomonas bacteria
282
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
283
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
284
What is the newest tool for genome editing?
CRISPR/Cas9
285
What does CRISPR/Cas9 do naturally?
Acts as a system to protect bacteria from bacteriophages
286
What is the difference between CRISPR/Cas9 and ZFNs/TALENs?
CRISPR/Cas9 does not rely on DNA-binding protein motifs
287
What does CRISPR stand for?
Clustered regulatory interspaced short palindromic repeats
288
What does Cas9 stand for?
CRISPR-associated enzyme
289
What does CRISPR/Cas9 do?
It can recognize DNA sequences by producing guide RNA molecules which can bind to complementary sequences
290
How can researchers modify the DNA sequence to be targeted by CRISPR/Cas9?
By changing the guide RNA sequence
291
How may improved functionality arise in a protein?
By adding more cysteine amino acids
292
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
293
What is an example of adding cysteine to stabilize a protein?
T4 lysozyme
294
What does T4 lysozyme do?
A bacterial cell wall degrading enzyme
295
Where was T4 lysozyme originally isolated from?
Phages
296
Where is T4 lysozyme used currently?
As a food preservative, and in infant formula
297
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
298
What is directed enzyme development?
Applying rounds of random mutagenesis and selection to achieve stepwise desired changes in a gene of interest
299
What does glycerol dehydrogenase do?
Catalyzes the formation of dihydroxyacetone
300
What does dihydroxyacetone do?
It is the main ingredient in sunless tanning products
301
What is error-prone PCR?
A method to produce variants of a particular enzyme
302
How does error-prone PCR work?
PCR is carried out under conditions with a very high error rate
303
How can the efficiency of directed enzyme development be increased?
Through DNA shuffling
304
What is the process for DNA shuffling?
Genes are digested and recombined in PCR to produce fusion, chimeric genes
305
How is DNA cleaved in DNA shuffling?
Through the enzyme DNase I
306
Why is DNA shuffling advanatgeous?
It can combine multiple beneficial variations into a single clone to create dramatically improved phenotypes
307
In microbial biotechnology, what is production limited by?
The genetic and physiological characteristics of the microbial strains used
308
How can the genetic and physiological characteristics of microbial strains be improved?
By making changes to the metabolic characteristics of the strain
309
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
310
How can high cell density of E. coli be achieved during fermentation?
To divert the precursor pyruvate away from the acetate pathway
311
How come the acetate pathway should be averted when growing E. coli for fermentation?
Lactate (a waste product) inhibits cell growth and protein production
312
How is the acetate pathway diverted in E. coli fermentation?
By using acetolactate synthase
313
What does acetolactase synthase do?
Converts pyruvate to acetoin, which is less inhibitory
314
What genes can be used to increase penicillin production?
cefEF and cefG
315
What does cefEF and cefG do?
They are involved in the biosynthesis of cephalosporin
316
What is needed (generally) for an expression vector to function?
The foreign (eukaryal) gene must have the correct components for bacterial transcription and translation
317
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
318
What is the importance of the Shine-Dalgarno sequence?
Bacterial ribosomes recognize this sequence on the mRNA to initiate translation
319
What is glycosylation?
The addition of complex sugar molecules to polypeptides
320
What is the issue with glycosylation?
Many animal proteins have glycosylation, but bacteria are not capable of doing this
321
What is the purpose of glycosylation?
It is needed for proper folding of the protein
322
How are many mammalian proteins glycosylated?
With sialic acid
323
How are many bacterial and fungal proteins glycosylated?
With mannose
324
Which amino acids are commonly glycosylated?
Serine and threonine
325
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
326
Besides glycosylation, what may be a problem for bacterial hosts when making eukaryal proteins?
Disulfide bonds
327
What is used to produce recombinant proteins in insect hosts?
Modified insect baculoviruses
328
Which cell lines can be used for recombinant protein production?
Chinese hamster ovary cells, and HeLa cells
329
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
330
True or false: a Shine-Dalgarno sequence is needed in a eukaryal expression vector
False: eukaryal cells do not use the Shine-Dalgarno sequence
331
What is synthetic biology?
Constructing novel biological systems from constituent parts
332
How much can 1kb of dsDNA be synthesized for?
$250
333
What is the tricky part of synthetic biology?
Making the synthetic DNA functional
334
What must DNA do to be functional?
Undergo replication, transcription, and translation
335
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
336
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
337
What is xenobiology?
The development of novel biological systems through expansion of the genetic code and incorporation of novel amino acids into proteins
338
How can xenobiology be useful?
It can enhance protein engineering and pathogen resistance in microorganisms
339
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
340
What are some examples of biological parts?
Enzyme-encoding genes, regulatory DNA sequences, or genes encoding regulatory proteins
341
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
342
What does the concept of biological parts have its roots in?
Engineering disciplines
343
What is the issue with biology and standards?
Biology does not adhere to any standards, unlike other engineering parts
344
What is the purpose of the Registry of Standard Biological Parts?
To help give biological parts some standard, similar to other engineering disciplines
345
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
346
How is each part in the Registry of Standard Biological Parts stored?
In a BioBrick
347
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
348
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
349
What makes it possible for eventual designing of plasmids by BioBricks?
The lowering cost of DNA synthesis
350
What does HMG stand for?
Hydroxymethylglutaryl
351
Who discovered the first statin?
Akira Endo
352
What did Akira Endo do?
Discover the first statin
353
If insulin is decreased, what happens to the blood sugar levels?
They increase
354
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
355
What is the advantage of using human insulin in bacteria over using pig or cattle insulin?
Human insulin is safer and more plentiful
356
What are some examples of fossil fuels?
Coal, oil, and natural gas
357
What are some examples of greenhouse gases?
CO2 (carbon dioxide) and N2O (nitrous oxide)
358
What products can be made from crude petroleum?
Gasoline, kerosene, wax, and asphalt
359
What does feedstock mean?
Raw starting material
360
What crops are specifically grown as biological feedstock?
Perennial grasses (Miscanthus), switchgrass, and poplar trees
361
How does distillation of alcohol work?
The culture is heated to evaporate the alcohol, which condenses around cooling coils. It can then be collected
362
In Brazil, what is the major source of ethanol?
Sugarcane
363
What problems are there with ethanol as a biofuel?
It only has 70% of the energy of gasoline, and it is more corrosive
364
What may be a better alternative to ethanol as a biofuel?
Butanol
365
What is butanol currently used for?
Latex, enamels, lacquers (make plastics flexible), and solvents
366
What are plastics?
Versatile polymers with a wide range of physical characteristics
367
What microbe was used for butanol fermentation?
Clostridium acetobutylicum
368
What is acetone used for?
Making cordite (the propellant used in military ammunition)
369
What are the two phases of butanol production?
1. CO2 was bubbled to exclude O2 and produce acids (acetate, butyrate, hydrogen, and CO2)
2. Acids were used to produce butanol, acetone, and ethanol
370
What was the typical yield of butanol after 60 hr?
12-20 g/L of solvent
371
How can PHB and PHA be altered for different properties?
By altering the length of the polymer, or the nature of the monomeric constituents
372
What is the key enzyme in PHA synthesis?
PHA synthase
373
What is the key enzyme in PHA degradation?
PHA depolymerase
374
What are the main goals of PHA bioplastics research?
1. Lower the cost of production to make it competitive
| 2. Produce plastics with properties appropriate for specific uses
375
Which specialized devices can be made with PHA and still be cost-competative?
Medical devices such as sutures, vascular stents, and tissue engineering scaffolds
376
Why would researchers want plants to produce PHAs?
They can use the sun's energy to make PHAs
377
What are some examples of enzymes used in white biotechnology?
Lipases, proteases, glycosidases, hydroxylases, nitrilases, acylases, and amidases
378
How is glucose isomerase used in soft drinks?
It adjusts the ration of glucose to fructose
379
What bacteria is glucose isomerase purified from?
Streptomyces sp.
380
Besides the laundry industry, what are some other key markets of enzymes in white biotechnology?
Baking, beverage, and dairy
381
How does a vitamin B12 deficiency occur?
Malabsorption in the gut (Crohn's disease, inflammatory bowel syndrome)
382
Which fungi are used to synthesize vitamin B2?
Ashbya gossypii and Eremothecium ashbyii
383
What is aspartame composed of?
L-aspartic acid and L-phenylalanine
384
Which microbes are used to synthesize aspartame?
Bacillus flavum (L-aspartic acid) and Clostridium glutamicum (L-phenylalanine)
385
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
386
Which microbe is used to synthesize L-lysine?
Corynebacterium glutamicum
387
What is the key feedback enzyme in lysine production?
Aspartate kinase
388
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
389
How does lysine interact with aspartate kinase?
Through negative feedback
390
What is used to select a mutant capable of high production of a product?
An antimetabolite
391
What is an antimetabolite?
A compound that closely resembles the structure of a natural compound
392
What is the antimetabolite for lysine?
AEC
393
What dies AEC stand for?
S-2-aminoethylcysteine
394
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
395
What are some common traits introduced into plants in green biotechnology?
Taste, yield, nutritional content, pest and pathogen resistance, and shelf life
396
Which researchers first discovered agrobacterium transformation?
Erwin Smith and C. O. Townsend
397
How was conjugation mediated by agrobacterium?
Through opines
398
What are opines and what do they do?
Amino acid-like compounds that aid in conjugation in agrobacterium
399
What does tDNA stand for?
Transfer DNA
400
How does tDNA aid in tumor formation in plants?
The tDNA from pTi is integrated into the plant genome
401
How does tDNA cause tumors in plants?
It produces opines and plant hormones (auxins, cytokinins) that promote plant cell growth
402
What is the advantage of infecting plants with tDNA for agrobacterium?
They can feed on the opines produced through opine catabolism genes on pTi
403
How is tDNA transfered?
Through the vir genes on the pTi plasmid
404
What is another name for glyphosate?
N-phosphonomethyl-glycine
405
What is glyphosate derived from?
Glycine
406
What does EPSP stand for?
5-enolpyruvylshikimate-3-phosphate
407
What is the problem with glyphosate for heribicides?
It kills most crop plants too
408
What is the significance of CTP?
The plant EPSP synthase enzymes localize to the chloroplasts
409
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)
410
What are the problems with most insecticides?
They are potent neurotoxins that can have detrimental effects on humans, livestock, wildlife, and non-pest insects
411
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
412
How does BT toxin become activated in the gut?
By dissolving in the alkaline conditions, and being digested by proteases
413
When was BT toxin first used as an insecticide?
1920s
414
In what bacterium are BT toxins expressed in now?
Pseudomonas fluorescens
