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BIO 315 > Module 8 - Microbial Biotechnology > Flashcards

Module 8 - Microbial Biotechnology Flashcards

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1
Q

What is biotechnology?

A

The exploitation of biological processes for industrial and other purposes

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2
Q

What are some common methods of biotechnology?

A

Genetic manipulation of microbes for production of proteins, antibiotics, hormones, and many other products

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3
Q

What has led to the development of molecular biology tools?

A

The understanding of genetic machinery of microbes

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4
Q

What can molecular biology tools be used for in biotechnology?

A

Used to develop microbes that can produce desired products

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5
Q

What is the intention of biotechnology?

A

To improve quality of human life

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6
Q

How has the biotechnology field changed over time?

A

It has grown very fast, and affecting almost all aspects of life

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7
Q

How come biotechnology has become widespread?

A

Due to the molecular biology tools developed

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8
Q

What are the three groups of biotechnology?

A

Red, white, and green

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9
Q

What is red biotechnology?

A

Medical applications

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10
Q

What is white biotechnology?

A

Industrial applications

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11
Q

What is green biotechnology?

A

Agricultural applications

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12
Q

What is bioprospecting?

A

Searching for useful new microbes to cultivate and add to collections

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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

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14
Q

What is metabolic/biochemical fermentation?

A

Catabolic reactants that produce ATP without oxygen

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15
Q

What is industrial fermentation?

A

The controlled and regulated aerobic/anaerobic culture of microbes to produce desired products

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16
Q

What is an example of biochemical fermentation?

A

Production of ethanol through yeast

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17
Q

What is the different between metabolic and industrial fermentation?

A

Metabolic fermentation has no oxygen, while industrial fermentation often uses oxygen

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18
Q

How is the majority of industrial fermentation carried out?

A

In the presence of oxygen

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19
Q

What is an example of industrial fermentation?

A

Large scale culture of E. coli for recombinant proteins

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20
Q

For the E. coli in industrial fermentation, what reaction is carried out?

A

Cellular respiration (in the presence of oxygen)

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21
Q

Where does industrial fermentation take place?

A

In bioreactors

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22
Q

What is another name for a bioreactor?

A

A fermenter

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23
Q

What do bioreactors do?

A

Control environmental conditions (nutrients, oxygen, pH, temperature) for industrial fermentation

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24
Q

What are the two basic types of bioreactors?

A

Fed-batch reactors and chemostats

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25
Q

How does a fed-batch reactor work?

A

High cell density is maintained by providing culture with growth limiting nutrients over time

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26
Q

Why does a fed-batch reactor provide growth limiting nutrients over time?

A

It prevents the production of non-desired side products

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27
Q

What is another name for a chemostat?

A

A continuous bioreactor

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28
Q

How does a chemostat work?

A

As new medium is added, the same amount of culture is removed

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29
Q

Why does a chemostats remove the same amount of culture as new medium is added?

A

It creates a precisely controlled constant growth rate of microbes

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30
Q

What does the choice of bioreactor depend on?

A

The type of microbe used and the nature of the desired end product

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31
Q

In practice, which bioreactor is more dependable and reproducible?

A

Fed-batch reactors

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32
Q

How do metabolites produced by cells depend on?

A

The growth phase of the microbes

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33
Q

What is the typical growth curve of a microorganism?

A

Lag phase, exponential phase, stationary phase, and death phase

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34
Q

To obtain the optimal production of a desired product, what might a researcher have to do?

A

Maintain the culture in a particular growth phase

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35
Q

How is the production of ethanol maximized in yeast?

A

They are maintained in the exponential phase with low oxygen and with glucose

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36
Q

For yeast growing under anoxic conditions, what is ethanol?

A

A primary metabolite

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37
Q

What is a primary metabolite?

A

The product of a metabolic process required for growth

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38
Q

What is a secondary metabolite?

A

A product not required for microbial growth

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39
Q

When is a secondary metabolite often produced?

A

During the stationary phase

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40
Q

What is an example of a secondary metabolite?

A

Antibiotics

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41
Q

When is a primary metabolite often produced?

A

During the exponential phase

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42
Q

What can genetic alterations in a microbe lead to (in biotechnology)?

A

Increased production of desired metabolites

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43
Q

What methods are used to mutate strains?

A

Random mutagenesis and site directed mutagenesis

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44
Q

How does random mutagenesis work?

A

Strains are exposed to mutagenic chemicals, and screened for a desired phenotype

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45
Q

What agents are used in random mutagenesis?

A

X-rays, UV light, or DNA damaging chemicals

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46
Q

What phenotypes would be screened for in random mutagenesis?

A

The production of an enzyme

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47
Q

What are the drawbacks of random mutagenesis?

A

The resulting mutations are undefined, they may be detrimental, and the screening can be difficult, labor-intensive, or costly

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48
Q

What is site directed mutagenesis?

A

A method to make specific mutations at specific known sites within a DNA molecule

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49
Q

What are the two types of site directed mutagenesis?

A

Oligonucleotide site-directed mutagenesis or PCR site-directed mutagenesis

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50
Q

What does oligonucleotide site-directed mutagenesis require?

A

Cloning of DNA in a vector that produces single stranded molecules

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51
Q

How does oligonucleotide site-directed mutagenesis work?

A

A complementary oligonucleotide with a desired sequence change is used for DNA synthesis in the presence of DNA polymerase

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52
Q

What happens after oligonucleotide site-directed mutagenesis?

A

Plasmids carrying the desired mutation can be recovered after transformation of E. coli

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53
Q

What does the oligonucleotide acts as in site-directed mutagenesis?

A

A primer for the DNA polymerase to continue on the plasmid

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54
Q

What does PCR site-directed mutagenesis require?

A

Complementary primers with the desired mutation

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55
Q

How does PCR site-directed mutagenesis work?

A

The complementary primers with the desired mutation will be used in a PCR reaction to create the mutation

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56
Q

What happens after PCR site-directed mutagenesis?

A

The PCR product is digested with Dpn1

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57
Q

What is Dpn1?

A

A restriction enzyme

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58
Q

What does Dpn1 do?

A

Cleaves the specific DNA site only when methylated during propagation of E. coli

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59
Q

What is the significance of Dpn1?

A

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

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60
Q

What DNA does Dpn1 cleave?

A

The original DNA template in the E. coli cell (methylated)

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61
Q

What DNA does Dpn1 not cleave?

A

The mutated DNA template outside the E. coli cell (unmethylated)

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62
Q

What is the consequence of Dpn1 cleavage?

A

The desired product will be enriched

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63
Q

How can the presence of a correct mutation be verified?

A

By DNA sequencing

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64
Q

How much of the total cell protein in E. coli is the most abundant native protein?

A

~2%

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65
Q

What do expression vectors do?

A

Produce recombinant proteins to higher levels

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66
Q

How high can expression vectors drive the production of recombinant proteins?

A

~20%

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67
Q

How can recombinant proteins be produced?

A

Through expression vectors

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68
Q

What are some examples of recombinant proteins produced by expression vectors?

A

Insulin, human growth hormone, and antiviral interferons

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69
Q

What are the parts of an expression vector?

A

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

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70
Q

What is the significance of the promoters in an expression vector?

A

They drive high levels of transcription

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71
Q

What is the significance of the operator in an expression vector?

A

Regulate the level of transcription

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72
Q

What is the significance of the ribosome binding site in an expression vector?

A

It helps with proper translation

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73
Q

What is needed in a ribosome binding site for an expression vector?

A

A Shine-Dalgarno sequence

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74
Q

What is the significance of the start and stop codons in an expression vector?

A

Used in translation to make the polypeptide

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75
Q

What is the significance of the transcriptional terminator sequences in an expression vector?

A

To end transcription

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76
Q

What is the sequence of the start codon?

A

ATG

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77
Q

How are fusion proteins produced?

A

Through expression vectors

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78
Q

What are fusion proteins?

A

Proteins that contain two or more domains of other proteins

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79
Q

What is another name for fusion proteins?

A

Tagged proteins

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80
Q

What do expression vectors designed for expressing fusion proteins have?

A

A built in sequence for a peptide tag fused to the coding sequence of the protein of interest

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81
Q

What may tags be used for (in fusion proteins)?

A

For purification

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82
Q

What is an affinity tag?

A

A tag that facilitates purification

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83
Q

What are some examples of affinity tags?

A

Protein A, His tags, MBP, GST, and strep-tag

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84
Q

What does His tag stand for?

A

Histidine amino acids

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85
Q

Wat does MBP stand for?

A

Maltose-binding protein

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86
Q

What does GST stand for?

A

Glutathione S-transferase

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87
Q

What is the receptor for protein A?

A

Anitbodies

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88
Q

What is the affinity tag for antibodies?

A

Protein A

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89
Q

What is the receptor for His tags?

A

Ni2+

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90
Q

What is the affinity tag for Ni2+?

A

His tags

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91
Q

What is the receptor for MBP?

A

Maltose

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92
Q

What is the affinity tag for maltose?

A

MBP

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93
Q

What is the receptor for GST?

A

Glutathion

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94
Q

What is the affinity tag for glutathion?

A

GST

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95
Q

What is the receptor for Strep-tag?

A

Streptavidin

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96
Q

What is the affinity tag for streptavidin?

A

Strep-tag

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97
Q

What are the major uses of microbes in red biotechnology?

A

Production of secondary metabolites with therapeutic properties, and production of recombinant human proteins

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98
Q

True or false: antibiotics are the only secondary metabolite that have therapeutic effects

A

False: other metabolites have been found

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99
Q

True or false: all therapeutic agents are from microbes

A

False: some are from plants

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100
Q

What is an example of a therapeutic agent derived from plants?

A

The antimalarial compound artemisinin

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101
Q

What are antibiotics?

A

Chemicals that kill or inhibit growth of bacteria

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102
Q

Where are antibiotics produced in nature?

A

By soil bacteria and fungi

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103
Q

What are antibiotics commonly used for?

A

Treating bacterial infections

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104
Q

How come antibiotcs gives microbes an advanatge?

A

They kill off the competition for limited resources

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105
Q

What was the first antibiotic discovered?

A

Penicillin

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106
Q

What organism produces penicillin?

A

Penicillium fungi

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107
Q

What antibiotics were discovered in the early 1940s?

A

Streptomycin and actinomycin

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108
Q

Where was streptomycin and actinomycin found?

A

In culture filtrates of actinomyces and saprophytic filamentous soil bacteria

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109
Q

How are many of the commercially available antibiotics made?

A

Through Streptomyces bacteria

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110
Q

True or false: all secondary metabolites in red biotechnology are antimicrobial

A

False: some can help in biochemical reactions

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111
Q

What do statins do?

A

They inhibit cholesterol synthesis

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112
Q

What organism produces statins?

A

Fungi

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113
Q

How do statins work?

A

They block the active site of HMG-CoA reductase

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114
Q

What does HMG-CoA reductase do?

A

It is important in cholesterol synthesis

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115
Q

What are some fungi that produce statins?

A

Penicillium and Aspergillus

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116
Q

What has lowered the production costs for many drugs?

A

Improvements of strains and fermentation techniques

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117
Q

What is a type I interferon?

A

Interferons with antitumor and antiviral capacities

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118
Q

What are some examples of recombinant human proteins used in red biotechnology?

A

Type I interferons, factor 13A, and insulin

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119
Q

What is factor 13A, and what is it used for?

A

A blood coagulation factor that is used to treat hemophilia

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120
Q

Where is insulin naturally produced?

A

In the pancreas

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121
Q

What does insulin do?

A

Regulates blood sugar levels

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122
Q

How was insulin extracted before red biotechnology?

A

From pigs and cattle

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123
Q

Which is more cost-effective for insulin production: E. coli or yeast?

A

Yeast

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124
Q

How come yeast is more cost effective for insulin production?

A

No purification steps are needed

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125
Q

What needs to be done if insulin is made from E. coli?

A

The E. coli endotoxin must be removed

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126
Q

What are the major uses of microbes in white biotechnology?

A

To generate products in many sectors, such as chemicals, food, detergents, bioplastics, and biofuels

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127
Q

What is the goal of white biotechnology?

A

To reduce greenhouse gas emissions and move away from petrochemical based products

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128
Q

What is the basic principle of white biotechnology?

A

To use microbial conversion of low-cost biomass to products with higher value

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129
Q

What do oil refineries do?

A

Convert crude petroleum into a number of useable products

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130
Q

What is the problem with oil refineries?

A

They generate numerous pollutants

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131
Q

What is biorefinery?

A

The process of converting biomass feedstocks into a number of products (chemicals/energy) with fewer environmental concerns

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132
Q

What are some examples of biomass that is otherwise discarded?

A

Crop plants and forestry waste

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133
Q

What provides the starting material for fuel production?

A

Cellulose/hemicellulose

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134
Q

What can cellulose/hemicellulose lead to?

A

Fuel production (ethanol/butanol), biochemicals (succinic acid and acetic acid) and other biopolymers

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135
Q

What is cellulose?

A

A complex polymer of beta-1,4-linked glucose units

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136
Q

True or false: most microbes can break down cellulose

A

False: very few can break down cellulose

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137
Q

What needs to be done to cellulose before it can be used by microbes?

A

It needs to be degraded by physical processes or enzymes

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138
Q

What did original diesel engines run on?

A

Biodiesel produced from vegetable oil

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139
Q

How come there is a renewed interest for ethanol as a biofuel?

A

The environmental concerns with fossil fuels

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140
Q

How is the commercial production of ethanol carried out?

A

Specific strains of yeast (efficient production properties)

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141
Q

What is the maximum amount of alcohol an industrial yeast strain can tolerate?

A

~15%

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142
Q

How is alcohol concentration increased in white biotechnology?

A

Through distillation processes

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143
Q

What does the cost of alcohol production depend on?

A

The cost of biomass feedstock used

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144
Q

In North America, how is most of the ethanol produced?

A

From corn starch

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145
Q

What is done to the corn starch before being used for ethanol production?

A

It is broken down into glucose using amylase

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146
Q

What does amylase do?

A

Breaks down starch into glucose

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147
Q

True or false: engines would need major modifications to use ethanol

A

False: ethanol can be used in internal combustion engines with little modification

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148
Q

What is commercial ethanol commonly produced from?

A

Sugar and starch

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149
Q

True or false: ethanol can be produced from cellulose

A

False: this is still currently being developed

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150
Q

What is the key step in ethanol production?

A

The conversion of biomass into cellulose that can be fermented to produce alcohol

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151
Q

What are some (2) unintended consequences of ethanol fuel?

A
  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
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152
Q

True or false: most waste biomass from agriculture can be used as feedstock

A

True: this can be a way to make the waste more useful

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153
Q

What waste biomass from agriculture can be used as feedstock?

A

Ligno-cellulose

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154
Q

What is ligno-cellulose?

A

A complex of cellulose, hemicellulose, and lignin

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155
Q

True or false: ligno-cellulose can be degraded by microbes

A

False: since it is a plant product, it cannot be broken down by microbes

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156
Q

What does cellulase do?

A

Converts ligno-cellulose into sugars

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157
Q

How is ligno-cellulose broken down?

A

Through cellulase

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158
Q

How can cellulase be used to make ethanol?

A

It can break ligno-cellulose into sugars, which can then be converted into alcohol by yeast

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159
Q

What is lignin?

A

A biodegradation-resistant phenolic polymer that surrounds polysaccharides

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160
Q

What is hemicellulose?

A

A complex polymer of hexoses, pentoses, and sugar acids

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161
Q

True or false: plastic waste is a major environmental problem

A

True: it can damage the environment

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162
Q

How can bioplastics serve as a solution to the plastic problem?

A

They are biodegradable, while other plastics are not

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163
Q

What do Bacillus megaterium bacteria produce?

A

PHB

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164
Q

What does PHB stand for?

A

Polyhydroxybutyrate

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165
Q

What bacteria produces PHB?

A

Bacillus megaterium

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166
Q

Besides PHB, what is another bioplastic polymer?

A

PHA

167
Q

What does PHA stand for?

A

Polyhydroxyalkanoates

168
Q

What bacteria produces PHA?

A

Multiple different types

169
Q

How do bacteria use PHAs?

A

They are a carbon storage polymer for a source of energy in starvation

170
Q

What can you say about materials constructed from PHAs?

A

They are biodegradable

171
Q

Where have PHA synthesis genes been expressed?

A

In a number of plants

172
Q

Why would PHA synthesis genes be expressed in plants?

A

These transgenic plants could then produce bioplastics

173
Q

What is the current state of bioplastics research?

A

While the amount of production has been low, ongoing research is promising

174
Q

What are the future goals of bioplastics?

A

Target PHA synthesis to specific parts of the plant (seed, leaf, stem), or produce PHA as a coproduct of crops (corn)

175
Q

What does the production of many commercial products depend on?

A

The use of enzymes

176
Q

What are some commercial products that depend on enzymes?

A

Food, detergents, and paper

177
Q

Where do many of the enzymes for commercial products originate from?

A

Microbes

178
Q

What is special about microbial enzymes?

A

They have high specificity and high efficiency, and are biodegradable

179
Q

What does HFCS stand for?

A

High fructose corn syrup

180
Q

Where is HFCS commonly found?

A

Soft drinks

181
Q

How is HFCS made?

A

From amylase on corn starch

182
Q

Where does the amylase enzyme for HFCS come from?

A

Bacillus bacteria

183
Q

What is the advantage of HFCS?

A

It is much cheaper than sugar

184
Q

What do several laundry detergents use?

A

A mixture of enzymes like lipases, amylases, proteases, glycosidases, and oxidases

185
Q

What do the enzymes in laundry detergents do?

A

Work together to remove dirt and stains from fabrics

186
Q

What is the largest single market for microbial enzymes?

A

The detergent industry

187
Q

How do researchers try to find better enzymes for white biotechnology?

A

Through bioprospecting or improving existing enzymes to get better results

188
Q

What is done to processed foods during production?

A

They are commonly fortified by adding compounds like vitamins

189
Q

What is another name for vitamin B9?

A

Folic acid

190
Q

Why is vitamin B9 added to foods?

A

It prevents growth defects in fetuses

191
Q

What foods have added vitamin B9?

A

Breads and pastas

192
Q

How are most vitamins synthesized?

A

Chemically

193
Q

What vitamins are synthesized by microbes?

A

Vitamin B12 and Vitamin B2

194
Q

What is another name for vitamin B12?

A

Cyanocobalamin

195
Q

What is another name for vitamin B2?

A

Riboflavin

196
Q

True or false: plants produce vitamin B12

A

False: they do not produce vitamin B12

197
Q

How do animals get vitamin B12?

A

Through gut microbes

198
Q

What can a vitamin B12 deficiency lead to?

A

Illness

199
Q

True or false: vitamin B12 is produced chemically

A

False: it is complicated

200
Q

What strains of bacteria are used to produce vitamin B12?

A

Pseudomonas or other bacterial strains

201
Q

True or false: vitamin B2 is produced chemically

A

True: the synthesis is much simpler than vitamin B12

202
Q

Which is simpler to produce chemically: vitamin B12 or vitamin B2?

A

Vitamin B2

203
Q

Besides vitamins, what other food supplement is produced by microorganisms?

A

Amino acids

204
Q

How are amino acids used in white biotechnology?

A

They are used in food, animal feed, nutritional supplement, and production of synthetic chemicals

205
Q

What is L-glutamic acid used for?

A

A flavor enhancer

206
Q

What is D/L-methionine used for?

A

Animal feed supplements

207
Q

What is L-lysine used for?

A

Animal feed supplements

208
Q

Which amino acid is used as a flavor enhancer?

A

L-glutamic acid

209
Q

Which amino acid is used in animal feed supplements?

A

D/L-methionine and L-lysine

210
Q

What are the most common amino acids produced in white biotechnology?

A

L-lysine, D/L-methionine, and L-glutamic acid

211
Q

What is an advantage of microbial production of amino acids?

A

Stereospecificity

212
Q

Which types of amino acids are produced from microbes?

A

L-isomers (which can be used by the human body)

213
Q

Which types of amino acids does the human body use?

A

L-isomers (which can be produced by microbes)

214
Q

What does present day agriculture involve?

A

The use of large amounts of insecticides, herbicides, and synthetic fertilizers

215
Q

What is the problem with intensive agriculture?

A

It poses concerns for human health and the environment

216
Q

What are the major uses of microbes in green biotechnology?

A

Pesticide/herbicide production, synthetic fertilizers, and introducing desired genes into plants

217
Q

What is one of the most efficient ways to introduce DNA into plants?

A

Through transformation by Agrobacterium

218
Q

What does Agrobacterium tumefaciens do?

A

Causes crown gull disease (tumors in many plants)

219
Q

True or false: agrobacterium is needed for the initiation of the tumor

A

True: the bacterium was required for initiatino

220
Q

True or false: agrobacterium is needed for the tumor to grow

A

False: the tumors continued to grow in the absence of the bacteria

221
Q

How did Agrobacterium cause tumors in plants?

A

Through the pTi plasmid’s tDNA

222
Q

How can the pTi plasmid move to other bacteria?

A

Through conjugation

223
Q

How can genes be introduced into plants?

A

Through agrobacterium mediated plant transformation

224
Q

What is agrobacterium mediated plant transformation?

A

Introducing new genes in a plant through tDNA mediated DNA transfer

225
Q

What is an example of cross-kingdom transfer of DNA?

A

Agrobacterium mediated plant transformation

226
Q

True or false: agrobacterium can be used to edit all plants

A

False: some plants cannot be edited this way

227
Q

What plants cannot be edited through agrobacterium?

A

Wheat, barley, and rice

228
Q

Besides agrobacterium, what are two methods to introduce DNA into a plant?

A

Protoplast formation and biolistics

229
Q

What is protoplast formation?

A

The removal of the cell wall prior to DNA introduction

230
Q

What is biolistics?

A

Using metal fragments coated with DNA, which are fired into the plant cells

231
Q

What is Roundup?

A

A broad spectrum herbicide

232
Q

Wat is the active ingredient in Roundup?

A

Glyphosate

233
Q

True or false: plants can absorb glyphosate

A

True: plant tissues absorb glyphosate efficiently

234
Q

What does glyphosate do?

A

It is a specific inhibitor of EPSP synthase

235
Q

What does EPSP synthase do?

A

It is a key step in synthesizing aromatic amino acids

236
Q

Which amino acids are aromatic?

A

Phenylalanine, tyrosine, and tryptophan

237
Q

What is the effect of glyphosate on plants?

A

They cannot produce essential amino acids

238
Q

True or false: glyphosate is toxic to humans and other mammals

A

False: it is most likely not toxic

239
Q

How come glyphosate is not toxic to humans?

A

They don’t have the specific pathway (with EPSP synthase) to produce aromatic amino acids

240
Q

How were glyphosate resistant transgenic plants generated?

A

By expressing an agrobacterium species CP4 EPSP synthase gene in several plants

241
Q

What is special about CP4 EPSP synthase?

A

It is resistant to glyphosate

242
Q

Which plants have had glyphosate tolerance incorporated?

A

Corn, cotton, and soybean

243
Q

What is the advantage of having transgenic plants with CP4 EPSP synthase?

A

Farmers can use Roundup all year round

244
Q

Which plasmid was used to introduce CP4 EPSP synthase into the plants?

A

PV-GMGT04

245
Q

What does the PV-GMGT04 plasmid contain?

A

Two copies of EPSP synthase fused to CTP, plant virus promoters, and a selectable marker gene

246
Q

What does CTP stand for?

A

Petunia Chloroplast Transit peptide

247
Q

What is the selectable marker gene in PV-GMGT04?

A

uidA (beta-glucuronidase marker gene)

248
Q

How was PV-GMGT04 introduced into the plants?

A

Through biolistics by Monsanto scientists

249
Q

What significantly impacts the growth of plants?

A

Weeds and insects

250
Q

What bacteria produce BT toxin?

A

Bacillus thuringiensis

251
Q

What does Bacillus thuringiensis do?

A

It is a spore forming bacterium that produces intracellular protein crystals

252
Q

What is another name for BT toxin?

A

Cry proteins

253
Q

What does BT toxin do (generally)?

A

It has highly specific insecticidal activity against moths, butterflies, mosquitos, and beetle larvae

254
Q

After being ingested, where is BT toxin activated?

A

In the insect gut

255
Q

After activation, what does BT toxin do?

A

It binds to specific receptors on gut epithelial cells, producing pores in the cell membrane

256
Q

What does BT toxin producing pores do?

A

It disrupts the osmotic balance, and kills the insect

257
Q

Why would bacteria naturally produce BT toxin?

A

Killing these insects likely provided a source of nutrients for vegetative growth

258
Q

What was the name of the original corn?

A

Teosinte

259
Q

How did teosinte evolve into modern corn?

A

Farmers picked teosinte that produced larger ears of corn, grew better in local environments, or were resistant to diseases

260
Q

At the very least, what must microbes be to be useful in biotechnology?

A

They must replicate well under standard laboratory conditions

261
Q

How long does it take for microbes with superior qualities to be selected?

A

Years

262
Q

How long does it take for crops with superior qualities to be selected?

A

Decades

263
Q

What are culture collections?

A

Publicly available archives of isolated and characterized microbes

264
Q

What is found in a culture collection?

A

Freeze-dried, frozen, or otherwise otherwise preserved living samples of microbial communities

265
Q

What is one of the most important challenges in the scale-up from laboratory to industrial culture?

A

The transfer of oxygen and maintenance of adequate mixing at very large volumes

266
Q

What is an example of a growth limiting nutrient in a Fed-batch reactor?

A

A carbon source

267
Q

True or false: primary metabolites can be overproduced without affecting the culture

A

False: they often impeded growth

268
Q

True or false: secondary metabolites can be overproduced without affecting the culture

A

True: they can produce toxicity or negative feedback

269
Q

How come primary metabolites can affect the culture when overproduced?

A

They are intrinsically linked to energy-production pathways, which can inhibit growth

270
Q

What genetic alterations can be done to make a microbe more useful for biotechnology?

A
  1. Manipulate expression of genes for enzyme production
  2. Downregulate competing pathways
  3. Grow on a lower-cost carbon source
271
Q

What are some examples of DNA damaging chemicals?

A

Nitrosoguanidine and ethyl methanesulfonate

272
Q

How was random mutagenesis used in red biotechnology?

A

It was used to produce strains of Penicillium chrysogenum with higher production of antibiotics

273
Q

What Penicillium was first isolated by Alexander Fleming?

A

Penicillium notatum

274
Q

What Penicillium was used for random mutagenesis for penicillin production?

A

Penicillium chrysogenum

275
Q

What is genome editing?

A

A process of precisely modifying genomes directly

276
Q

What was used for the first genome editing?

A

ZFNs and TALENs

277
Q

What does ZFN stand for?

A

Zinc finger nucleases

278
Q

What does TALEN stand for?

A

Transcription activator-like effector nucleases

279
Q

What do ZFNs do?

A

They are well known motifs involved in recognizing and binding specific DNA sequences

280
Q

What do TALENs do?

A

They are DNA binding proteins found in Xanthomonas bacteria

281
Q

Where are TALENs found?

A

In Xanthomonas bacteria

282
Q

How can ZFNs and TALENs be used in genome editing?

A

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
Q

What is the difficulty of using ZFNs and TALENs in genome editing?

A

The design of the specificity of the DNA-binding domains is often tedious

284
Q

What is the newest tool for genome editing?

A

CRISPR/Cas9

285
Q

What does CRISPR/Cas9 do naturally?

A

Acts as a system to protect bacteria from bacteriophages

286
Q

What is the difference between CRISPR/Cas9 and ZFNs/TALENs?

A

CRISPR/Cas9 does not rely on DNA-binding protein motifs

287
Q

What does CRISPR stand for?

A

Clustered regulatory interspaced short palindromic repeats

288
Q

What does Cas9 stand for?

A

CRISPR-associated enzyme

289
Q

What does CRISPR/Cas9 do?

A

It can recognize DNA sequences by producing guide RNA molecules which can bind to complementary sequences

290
Q

How can researchers modify the DNA sequence to be targeted by CRISPR/Cas9?

A

By changing the guide RNA sequence

291
Q

How may improved functionality arise in a protein?

A

By adding more cysteine amino acids

292
Q

How come adding more cysteine amino acids may help improve functionality of the protein?

A

Disulfide bonds stabilize proteins, making them less susceptible to denaturing conditions like heat or pH

293
Q

What is an example of adding cysteine to stabilize a protein?

A

T4 lysozyme

294
Q

What does T4 lysozyme do?

A

A bacterial cell wall degrading enzyme

295
Q

Where was T4 lysozyme originally isolated from?

A

Phages

296
Q

Where is T4 lysozyme used currently?

A

As a food preservative, and in infant formula

297
Q

What does the successful alteration of a protein via site-directed mutagenesis require?

A

A fairly detailed knowledge of the three-dimensional structure of the protein

298
Q

What is directed enzyme development?

A

Applying rounds of random mutagenesis and selection to achieve stepwise desired changes in a gene of interest

299
Q

What does glycerol dehydrogenase do?

A

Catalyzes the formation of dihydroxyacetone

300
Q

What does dihydroxyacetone do?

A

It is the main ingredient in sunless tanning products

301
Q

What is error-prone PCR?

A

A method to produce variants of a particular enzyme

302
Q

How does error-prone PCR work?

A

PCR is carried out under conditions with a very high error rate

303
Q

How can the efficiency of directed enzyme development be increased?

A

Through DNA shuffling

304
Q

What is the process for DNA shuffling?

A

Genes are digested and recombined in PCR to produce fusion, chimeric genes

305
Q

How is DNA cleaved in DNA shuffling?

A

Through the enzyme DNase I

306
Q

Why is DNA shuffling advanatgeous?

A

It can combine multiple beneficial variations into a single clone to create dramatically improved phenotypes

307
Q

In microbial biotechnology, what is production limited by?

A

The genetic and physiological characteristics of the microbial strains used

308
Q

How can the genetic and physiological characteristics of microbial strains be improved?

A

By making changes to the metabolic characteristics of the strain

309
Q

What is an example of manipulating metabolic characteristics in microbial strains?

A

Genes can be introduced that allow the microbe to grow on a cheaper carbon source

310
Q

How can high cell density of E. coli be achieved during fermentation?

A

To divert the precursor pyruvate away from the acetate pathway

311
Q

How come the acetate pathway should be averted when growing E. coli for fermentation?

A

Lactate (a waste product) inhibits cell growth and protein production

312
Q

How is the acetate pathway diverted in E. coli fermentation?

A

By using acetolactate synthase

313
Q

What does acetolactase synthase do?

A

Converts pyruvate to acetoin, which is less inhibitory

314
Q

What genes can be used to increase penicillin production?

A

cefEF and cefG

315
Q

What does cefEF and cefG do?

A

They are involved in the biosynthesis of cephalosporin

316
Q

What is needed (generally) for an expression vector to function?

A

The foreign (eukaryal) gene must have the correct components for bacterial transcription and translation

317
Q

How can introns be removed from a gene?

A

By either artificial synthesis, or by using an mRNA transcript as the template for reverse transcriptase to make cDNA

318
Q

What is the importance of the Shine-Dalgarno sequence?

A

Bacterial ribosomes recognize this sequence on the mRNA to initiate translation

319
Q

What is glycosylation?

A

The addition of complex sugar molecules to polypeptides

320
Q

What is the issue with glycosylation?

A

Many animal proteins have glycosylation, but bacteria are not capable of doing this

321
Q

What is the purpose of glycosylation?

A

It is needed for proper folding of the protein

322
Q

How are many mammalian proteins glycosylated?

A

With sialic acid

323
Q

How are many bacterial and fungal proteins glycosylated?

A

With mannose

324
Q

Which amino acids are commonly glycosylated?

A

Serine and threonine

325
Q

What needs to be done if a eukaryal protein needs a specific glycosylation to function properly?

A

A eukaryal host (not a bacterial host) will be needed

326
Q

Besides glycosylation, what may be a problem for bacterial hosts when making eukaryal proteins?

A

Disulfide bonds

327
Q

What is used to produce recombinant proteins in insect hosts?

A

Modified insect baculoviruses

328
Q

Which cell lines can be used for recombinant protein production?

A

Chinese hamster ovary cells, and HeLa cells

329
Q

What is the disadvantage of eukaryal host lines for recombinant protein production?

A

They are more expensive and often do not produce as much recombinant protein

330
Q

True or false: a Shine-Dalgarno sequence is needed in a eukaryal expression vector

A

False: eukaryal cells do not use the Shine-Dalgarno sequence

331
Q

What is synthetic biology?

A

Constructing novel biological systems from constituent parts

332
Q

How much can 1kb of dsDNA be synthesized for?

A

$250

333
Q

What is the tricky part of synthetic biology?

A

Making the synthetic DNA functional

334
Q

What must DNA do to be functional?

A

Undergo replication, transcription, and translation

335
Q

What is needed to create a truly synthetic organism?

A

The entire microbial genome would need to be synthesized, introduced in a cell, replicate, and replace the preexisting host cell DNA

336
Q

What does the synthetic organism E. coli do in xenobiology?

A

It can code for phosphoserine, a novel amino acid not found in nature, with the UAG stop codon

337
Q

What is xenobiology?

A

The development of novel biological systems through expansion of the genetic code and incorporation of novel amino acids into proteins

338
Q

How can xenobiology be useful?

A

It can enhance protein engineering and pathogen resistance in microorganisms

339
Q

What needs to be done when considering the design of microbes for a specific function?

A

Genetic elements must be joined in different combinations and under appropriate regulation

340
Q

What are some examples of biological parts?

A

Enzyme-encoding genes, regulatory DNA sequences, or genes encoding regulatory proteins

341
Q

What is the goal with biological parts?

A

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
Q

What does the concept of biological parts have its roots in?

A

Engineering disciplines

343
Q

What is the issue with biology and standards?

A

Biology does not adhere to any standards, unlike other engineering parts

344
Q

What is the purpose of the Registry of Standard Biological Parts?

A

To help give biological parts some standard, similar to other engineering disciplines

345
Q

What does the Registry of Standard Biological Parts contain?

A

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
Q

How is each part in the Registry of Standard Biological Parts stored?

A

In a BioBrick

347
Q

What is a BioBrick?

A

A vector that is flanked by EcoRI and XbaI sites on one side, and SpeI and PstI sites on the other side

348
Q

Why is XbaI and SpeI used in Biobricks?

A

They have the same core 4-nucleotide base sequence, only differing in flanking nucleotides. Thus, they have compatible sticky ends

349
Q

What makes it possible for eventual designing of plasmids by BioBricks?

A

The lowering cost of DNA synthesis

350
Q

What does HMG stand for?

A

Hydroxymethylglutaryl

351
Q

Who discovered the first statin?

A

Akira Endo

352
Q

What did Akira Endo do?

A

Discover the first statin

353
Q

If insulin is decreased, what happens to the blood sugar levels?

A

They increase

354
Q

How is insulin created in E. coli?

A

Two cultures have the two different chains (alpha and beta), which are fused together through disulfide bonds later

355
Q

What is the advantage of using human insulin in bacteria over using pig or cattle insulin?

A

Human insulin is safer and more plentiful

356
Q

What are some examples of fossil fuels?

A

Coal, oil, and natural gas

357
Q

What are some examples of greenhouse gases?

A

CO2 (carbon dioxide) and N2O (nitrous oxide)

358
Q

What products can be made from crude petroleum?

A

Gasoline, kerosene, wax, and asphalt

359
Q

What does feedstock mean?

A

Raw starting material

360
Q

What crops are specifically grown as biological feedstock?

A

Perennial grasses (Miscanthus), switchgrass, and poplar trees

361
Q

How does distillation of alcohol work?

A

The culture is heated to evaporate the alcohol, which condenses around cooling coils. It can then be collected

362
Q

In Brazil, what is the major source of ethanol?

A

Sugarcane

363
Q

What problems are there with ethanol as a biofuel?

A

It only has 70% of the energy of gasoline, and it is more corrosive

364
Q

What may be a better alternative to ethanol as a biofuel?

A

Butanol

365
Q

What is butanol currently used for?

A

Latex, enamels, lacquers (make plastics flexible), and solvents

366
Q

What are plastics?

A

Versatile polymers with a wide range of physical characteristics

367
Q

What microbe was used for butanol fermentation?

A

Clostridium acetobutylicum

368
Q

What is acetone used for?

A

Making cordite (the propellant used in military ammunition)

369
Q

What are the two phases of butanol production?

A
  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
Q

What was the typical yield of butanol after 60 hr?

A

12-20 g/L of solvent

371
Q

How can PHB and PHA be altered for different properties?

A

By altering the length of the polymer, or the nature of the monomeric constituents

372
Q

What is the key enzyme in PHA synthesis?

A

PHA synthase

373
Q

What is the key enzyme in PHA degradation?

A

PHA depolymerase

374
Q

What are the main goals of PHA bioplastics research?

A
  1. Lower the cost of production to make it competitive

2. Produce plastics with properties appropriate for specific uses

375
Q

Which specialized devices can be made with PHA and still be cost-competative?

A

Medical devices such as sutures, vascular stents, and tissue engineering scaffolds

376
Q

Why would researchers want plants to produce PHAs?

A

They can use the sun’s energy to make PHAs

377
Q

What are some examples of enzymes used in white biotechnology?

A

Lipases, proteases, glycosidases, hydroxylases, nitrilases, acylases, and amidases

378
Q

How is glucose isomerase used in soft drinks?

A

It adjusts the ration of glucose to fructose

379
Q

What bacteria is glucose isomerase purified from?

A

Streptomyces sp.

380
Q

Besides the laundry industry, what are some other key markets of enzymes in white biotechnology?

A

Baking, beverage, and dairy

381
Q

How does a vitamin B12 deficiency occur?

A

Malabsorption in the gut (Crohn’s disease, inflammatory bowel syndrome)

382
Q

Which fungi are used to synthesize vitamin B2?

A

Ashbya gossypii and Eremothecium ashbyii

383
Q

What is aspartame composed of?

A

L-aspartic acid and L-phenylalanine

384
Q

Which microbes are used to synthesize aspartame?

A

Bacillus flavum (L-aspartic acid) and Clostridium glutamicum (L-phenylalanine)

385
Q

What are some features of microbial strains used for producing metabolites in white biotechnology?

A

They cannot survive and compete in their natural habitat because they are metabolically compromised

386
Q

Which microbe is used to synthesize L-lysine?

A

Corynebacterium glutamicum

387
Q

What is the key feedback enzyme in lysine production?

A

Aspartate kinase

388
Q

What would the features of aspartate kinase be for a good microbe in biotechnology?

A

One where the regulatory allosteric site is non-functional, but where the active site is functional

389
Q

How does lysine interact with aspartate kinase?

A

Through negative feedback

390
Q

What is used to select a mutant capable of high production of a product?

A

An antimetabolite

391
Q

What is an antimetabolite?

A

A compound that closely resembles the structure of a natural compound

392
Q

What is the antimetabolite for lysine?

A

AEC

393
Q

What dies AEC stand for?

A

S-2-aminoethylcysteine

394
Q

How is AEC used to select for mutants that can produce lysine?

A

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
Q

What are some common traits introduced into plants in green biotechnology?

A

Taste, yield, nutritional content, pest and pathogen resistance, and shelf life

396
Q

Which researchers first discovered agrobacterium transformation?

A

Erwin Smith and C. O. Townsend

397
Q

How was conjugation mediated by agrobacterium?

A

Through opines

398
Q

What are opines and what do they do?

A

Amino acid-like compounds that aid in conjugation in agrobacterium

399
Q

What does tDNA stand for?

A

Transfer DNA

400
Q

How does tDNA aid in tumor formation in plants?

A

The tDNA from pTi is integrated into the plant genome

401
Q

How does tDNA cause tumors in plants?

A

It produces opines and plant hormones (auxins, cytokinins) that promote plant cell growth

402
Q

What is the advantage of infecting plants with tDNA for agrobacterium?

A

They can feed on the opines produced through opine catabolism genes on pTi

403
Q

How is tDNA transfered?

A

Through the vir genes on the pTi plasmid

404
Q

What is another name for glyphosate?

A

N-phosphonomethyl-glycine

405
Q

What is glyphosate derived from?

A

Glycine

406
Q

What does EPSP stand for?

A

5-enolpyruvylshikimate-3-phosphate

407
Q

What is the problem with glyphosate for heribicides?

A

It kills most crop plants too

408
Q

What is the significance of CTP?

A

The plant EPSP synthase enzymes localize to the chloroplasts

409
Q

Besides glyphosate, what are some other herbicides?

A

Glufosinate (inhibitor of glutamine synthetase enzyme) and 2,4-dichlorophenoxyacetic acid (2,4-D) (synthetic version of auxin phytohormone)

410
Q

What are the problems with most insecticides?

A

They are potent neurotoxins that can have detrimental effects on humans, livestock, wildlife, and non-pest insects

411
Q

What are the scientific names for the organisms that BT toxin is effective against?

A

Lepidopteran (moths and butterflies), dipteran (flies and mosquitos), and coleopteran (beetle) larvae

412
Q

How does BT toxin become activated in the gut?

A

By dissolving in the alkaline conditions, and being digested by proteases

413
Q

When was BT toxin first used as an insecticide?

A

1920s

414
Q

In what bacterium are BT toxins expressed in now?

A

Pseudomonas fluorescens

BIO 315 (12 decks)

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