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BESTE SAMENVATTING BIOTECH OOIT.

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Biotechnology

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Vrije Universiteit Brussel (VUB)

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Biotechnology
Summary
Coronavirus Edition - 2020

, Biotechnology

Table of contents
CHAPTER 1 : THE CENTURY OF BIOTECHNOLOGY ....................................................... Error! Bookmark not defined.
1.1. What is biotechnology and what does it mean to you? ..............................................................5
1.2. Different kinds of Biotechnology............................................................................................. 11
1.3. Biological challenges of the 21st century ................................................................................. 16
1.4. The ‘Workforce’ in biotechnology ........................................................................................... 19
1.5. VIB (Vlaams interuniversitair instituut voor Biotechnologie) ..................................................... 22
CHAPTER 2: CELLULAR LIFE AND MOLECULES OF LIFE ............................................... Error! Bookmark not defined.
2.1. All cells are either prokaryote or eukaryote ............................................................................. 23
2.2. Cells come in many sizes and shapes............................................. Error! Bookmark not defined.
2.3. The prokaryotic cell ............................................................................................................... 27
2.4. The eukaryotic cell ................................................................................................................. 28
2.5. Prokaryotes and eukaryotes differ from each other in many ways ............................................ 31
2.6. Viruses.................................................................................................................................. 32
2.7. Molecules of Life .......................................................................... Error! Bookmark not defined.
a) Fatty acids, lipids and membranes ..................................................... Error! Bookmark not defined.
b) Sugars and carbohydrates ................................................................ Error! Bookmark not defined.
c) Amino acids and Proteins ................................................................. Error! Bookmark not defined.
d) Bases, Nucleosides, Nucleotides and Nucleic Acids ............................ Error! Bookmark not defined.
CHAPTER 3: RECOMBINANT DNA TECHNOLOGY AND G ENOMICS ........................................................................... 78
3.1. Introduction to Recombinant DNA Technology and DNA Cloning .............................................. 78
Restriction enzymes ............................................................................. Error! Bookmark not defined.
DNA ligase ..................................................................................................................................... 85
Plasmid DNAs................................................................................................................................. 87
Vectors .......................................................................................................................................... 87
Transformation of bacterial cells ..................................................................................................... 87
3.2. What Makes a Good Vector?.................................................................................................. 89
Selection........................................................................................................................................ 89
Practical Features of DNA Cloning Vectors ....................................................................................... 93
Types of vectors (not discussed in class) .......................................................................................... 93
3.3. How Do You Identify and Clone a Gene of Interest? .................................................................. 95
Creating DNA Libraries.................................................................................................................... 97
Types of Libraries ........................................................................................................................... 97

, Comparison of a genomic DNA library and a cDNA library ................................................................. 97
Library screening.......................................................................................................................... 106
Synthesis of oligonucleotides ........................................................................................................ 107
Polymerase chain reaction (PCR) ................................................................................................... 109
Cloning PCR products.................................................................................................................... 113
3.4. What Can You Do with a Cloned Gene? Applications of Recombinant DNA Technology............ 118
DNA sequencing........................................................................................................................... 119
Studying gene expression ............................................................................................................. 127
3.5. Genomics and Bioinformatics: Hot New Areas of Biotechnology ............................................. 133
Shot-gun sequencing .................................................................................................................... 133
The Human Genome Project ......................................................................................................... 133
Comparative genomics ................................................................................................................. 133
CHAPTER 4: PROTEINS AS PRODUCTS............................................................................................................. 134
4.1. Introduction to Proteins as Biotech Products ......................................................................... 134
4.2. Proteins as Biotechnology Products .............................................. Error! Bookmark not defined.
Medical applications..................................................................................................................... 136
Food processing ........................................................................................................................... 137
Textiles and clothing..................................................................................................................... 137
Detergents................................................................................................................................... 137
Paper manufacturing and recycling ............................................................................................... 137
Adhesives: natural glues ............................................................................................................... 137
Bioremediation: treating pollution with proteins............................................................................ 137
4.3. Protein Structures................................................................................................................ 138
Structural arrangement ................................................................................................................ 140
Protein folding ............................................................................................................................. 145
Glycosylation ....................................................................................... Error! Bookmark not defined.
Protein engineering...................................................................................................................... 147
4.4. Protein Production............................................................................................................... 148
Protein Expression: upstream processing ....................................................................................... 149
Protein Purification Methods: downstream processing ................................................................... 151
Verification .......................................................................................... Error! Bookmark not defined.
Preserving Proteins ...................................................................................................................... 157
Scale-Up of Protein Purification............................................................. Error! Bookmark not defined.
Postpurification Analysis Methods................................................................................................. 157
4.5. Proteomics.......................................................................................................................... 158
CHAPTER 5: MICROBIAL BIOTECHNOLOGY ....................................................................................................... 159
5.1. The Structure of Microbes .................................................................................................... 159

, Structural Features of Bacteria ...................................................................................................... 159
Yeast ........................................................................................................................................... 161
5.2. Microorganisms as Tools...................................................................................................... 162
Microbial Enzymes ....................................................................................................................... 162
Bacterial transformation............................................................................................................... 162
Cloning and expression techniques................................................................................................ 163
5.3. Using Microbes for a Variety of Everyday Applications ................... Error! Bookmark not defined.
Food products.............................................................................................................................. 165
Therapeutic proteins .................................................................................................................... 166
Field applications ................................................................................. Error! Bookmark not defined.
5.4. Vaccines.............................................................................................................................. 169
Immune system and antibodies..................................................................................................... 169
Types of vaccines: how are vaccines made? ................................................................................... 172
Bacterial and viral targets for vaccines ........................................................................................... 173
5.5. Microbial Genomes.............................................................................................................. 176
Why sequence microbial genomes?............................................................................................... 177
Viral genomics.............................................................................................................................. 176
5.6. Microbial Diagnostics ........................................................................................................... 178
Bacterial Detection Strategies ....................................................................................................... 178
Microarrays for tracking contagious diseases ................................................................................. 179
5.7. Combating Bioterrorism....................................................................................................... 179
Microbes as bioweapons .............................................................................................................. 179
5.8. Synthetic Biology ......................................................................... Error! Bookmark not defined.
CHAPTER 6: ANIMAL BIOTECHNOLOGY........................................................................................................... 182
6.1. Introduction to Animal Biotechnology................................................................................... 193
6.2. Animals in Research............................................................................................................. 193
Why use animals in research? ....................................................................................................... 193
Regulations in animal research.............................................................. Error! Bookmark not defined.
Types of animals used in research ................................................................................................. 194
Alternatives to the use of animals ................................................................................................. 195
Regulation of animal research ....................................................................................................... 195
Veterinary Medicine as Clinical Trials ............................................................................................. 196
6.3. Clones.................................................................................................................................. 196
Creating Dolly: a breakthrough in cloning .............................................. Error! Bookmark not defined.
Limits to cloning ........................................................................................................................... 197
The Future of Cloning ................................................................................................................... 198
6.4. Transgenic Animals .............................................................................................................. 199

, Transgenic techniques .................................................................................................................. 199
Increasing agricultural production with transgenics ........................................................................ 200
Transgenic Animals as Bioreactors................................................................................................. 201
Knockouts: A Special Case of Transgenics ...................................................................................... 202
6.5. Producing Human Antibodies in Animals ............................................................................... 204
Monoclonal antibodies ................................................................................................................. 204
CHAPTER 7: MEDICAL BIOTECHNOLOGY.......................................................................................................... 206
7.1. The Power of Molecular Biology: Detecting and Diagnosing Human Disease Conditions........... 206
Models of Human Disease............................................................................................................. 206
Biomarkers for Disease Detection.................................................................................................. 206
The Human Genome Project Has Revealed Disease Genes on All Human Chromosomes................... 208
Detecting Genetic Diseases: Testing for chromosome abnormalities ............................................... 209
7.2. Medical Products and Applications of Biotechnology ............................................................. 213
The search for new medicines and drugs ....................................................................................... 214
Vaccines and Therapeutic Antibodies .................................................... Error! Bookmark not defined.
7.3. Gene Therapy.............................................................................. Error! Bookmark not defined.
How is it done? ............................................................................................................................ 230
Curing genetic diseases: targets for gene therapy................................... Error! Bookmark not defined.
Challenges Facing Gene Therapy ................................................................................................... 235
7.4. The Potential of Regenerative Medicine ........................................ Error! Bookmark not defined.
Cell and Tissue Transplantation ............................................................. Error! Bookmark not defined.
Tissue Engineering................................................................................ Error! Bookmark not defined.
Stem Cell Technologies ......................................................................... Error! Bookmark not defined.
Cloning ................................................................................................ Error! Bookmark not defined.
Embryonic stem cell and therapeutic cloning regulations in U.S .............. Error! Bookmark not defined.
EXAMPLE OF 1ST QUESTION AT EXAMS .......................................................................................................... 237

,CHAPTER 1 : THE CENTURY OF BIOTECHNOLOGY
Biotechnology is everywhere. A biotechnological product is for example wine, beer, sour cream,
yoghurt, cheese, milk, vegetable oils, antibiotics, … But biotechnology is sometimes considered as
something bad, GMO (genetic modified organisms) – opinions differ. Also cloning and stem cell
research is highly controversial – in the US it is not allowed.

Learning objectives of this class:

− Emergence of biotechnology: where does biotechnology come from?
− Define biotechnology & understand the many scientific aspects that contribute to it
− Know some biotech applications and examples of biotech derived products
− Advantages of biotechnology
− Novel challenges in biotechnology
− The controversial matters in biotechnology
− The ‘workforce’ in biotechnology

1.1 What is biotechnology and what does it mean to you?

People started to investigate things because they were curious. And very soon knowledge started to
expand so you could not be a master in everything anymore. So, they divided sciences in different
categories: physics, biology and chemistry. The first challenges of chemistry were how to make gold
(convert iron into gold) and how to live longer (eternal life / stay young forever; antiwrinkle crème, …).
Later, these sciences started to merge: biophysics 1 (physics & biology), physic-chemistry (physics &
chemistry) and molecular biology (if you started as a biologist) or biochemistry (if you started as a
chemist). These sciences can be exploited, and several professions emerged. The biotechnologist came
in the 1970’s, where biochemistry and molecular biology are the fundamentals.

1 You can for example learn how there are very tall trees and how the leaflets get water from the roots 30 meters
lower.

5

,Broad definition of biotechnology: the science of using living organisms, or the products of living
organisms, for human benefit (or to benefit human surroundings) 2, that is, to make a product or solve
a problem (or by providing a relieve to).

Forecasting the future:
- 2020: Biotech companies generate global revenues of $163 billion.
- = 20% of the revenues generated by the pharmaceutical market
- Current annual growth rate of biotech companies is > 8%
- (conventional pharma companies round 4%)

Historical 3 examples of biotechnology 4 are:
− Fermentation: to make breads, cheeses yoghurts, and alcoholic beverages such as beer and wine.
During fermentation, some strains of yeast decompose sugars to derive energy, and in the
process they produce ethanol (alcohol) as a waste product. When bread dough is being made,
yeast (Saccharomyces cerevisiae, commonly called baker’s yeast) is added to make the dough
rise. This occurs because the yeast ferments sugar-releasing carbon dioxide, which causes the
dough to rise and creates holes in the bread. Alcohol produced by the yeast evaporates when the
bread is cooked.

// The reason for this was to stay healthy. Extra example, the water in the river can be filled with
pathogens/bad bacteria from dead animals. By taking the water, putting sugars to it and letting it ferment,
they are producing ethanol. But ethanol is a toxic compound, so in the end the yeast that are pr oducing
the ethanol are getting killed by the ethanol. (In Germany, there was an outbreak of contaminate d
cucumbers in 2011, where 50 people died.) //

− Selective breeding: to improve production of crops and livestock used for food purposes. In SB
organisms with desirable features are purposely mated to produce offspring with the same
desirable characteristics. It is an old example that is still common today. For example,
crossbreeding plants that produce the largest, sweetest, and most tender ears of corn (or chicken
breast) is a good way for farmers to maximize their land to produce the most desirable crops.

Important example: zebrafish “Danio rerio” have normally black – white stripes. But they isolated
a mutant that lacked reflective pigment, and a mutant that lacked black pigment. Therefore, they
crossed these two and selected the offspring that is completely transparent. Making that you can
see all the organs of these fish, proven to be valuable for studying how cancer cells spread:
scientists injected fluorescent tumor cells into the fish’s abdominal cavity and were able to track
the migration of those cells to specific locations in the body.

− Use of antibiotics: Antibiotics are substances produced by microorganisms (a product of fungi or
bacteria) that will inhibit the growth of other microorganisms.

2 Animals, plants, environment
3 Historical accounts have shown that people have been involved in biotechnology since about 2000 b.c.
4 Biotechnology does not mean hunting and gathering animals and plants for food; however, the domestication

of animals (to produce milk, eggs, etc.) such as sheep and cattle for use as livestock is a classic example of
biotechnology.

6

,Modern examples are:
- Use of antibiotics: Batch (large-scale) processes: —in which scientists can grow bacteria and other
cells in large amounts and harvest useful products in large batches—were developed to isolate
commercially important molecules from microorganisms (explained further in Chapter 4).

− Gene cloning: the ability to identify and reproduce a gene of interest, and genetic engineering,
manipulating the DNA of an organism. Through genetic engineering, scientists are able to combine
DNA from different sources. This process, called recombinant DNA technology, is used to produce
many proteins of medical importance, including insulin, human growth hormone, and blood-
clotting factors. Other examples are the development of disease-resistant plants, food crops that
produce greater yields, “golden rice” engineered to be more nutritious and genetically engineered
bacteria capable of degrading environmental pollutants. From its inception, recombinant DNA
technology has dominated many important areas of biotechnology, and many credit recombinant
DNA technology with starting modern biotechnology as an industry.

(They changed the name regularly because people thought this was a bad thing (cloning a new
Frankenstein or Hitler).

− ‘Human Genome Project’: was an international scientific research project with the primary goal of
identify all genes—the genome—contained in the DNA of human cells and to map their locations
to each of the 24 human chromosomes (chromosomes 1 to 22 and the X and Y chromosomes).

• Genomics (the study of genomes)
• Artificial or synthetic genomes
• Genome editing: CRISPR-Cas

Gene maps of chromosomes 13 and 21 resulting from the Human Genome Project

7

,Products of modern biotechnology
 Many of the most widely used products of biotechnology are proteins created by gene cloning,
referred to as recombinant proteins. For example, the majority of these proteins are produced
from human genes inserted into bacteria to make the recombinant proteins used to treat human
disease conditions (see picture next page). (see CH3)

Table 1.1.: examples of proteins manufactured from cloned genes
PRODUCT APPLICATION
Blood factor VIII (clotting factor) Used to treat hemophilia
Epidermal growth factor Used to stimulate antibody production in patients with
immune system disorders
5
Growth hormone Used to correct pituitary (slijmafscheidende)
deficiencies and short stature in humans; other forms
used in cows to increase milk production
Insulin6 Used to treat diabetes mellitus
Interferons Used to treat cancer and viral infections
Interleukins Used to treat cancer and stimulate antibody production
Monoclonal antibodies Used to diagnose and treat a variety of diseases
including cancers
7
Tissue plasminogen activator (TPA) Used to treat heart attacks and stroke

 Brief list of some of the top-selling biotechnology drugs (each with worldwide sales over $5
billion) and the companies that developed them.

Table 1.2: TOP 10 BIOTECHNOLOGY DRUGS (Sales > $1 billion)

DRUG NAME DEVELOPER DRUG TYPE FUNCTION
Humira AbbVie Antibody (monoclonal) Rheumatoid arthritis, Crohn’s
disease, Ulcerative colitis
Harvoni Gilead Sciences Small molecule Hepatitis C
Rituxan Roche Antibody (monoclonal) Non-Hodgkin’s lymphoma
Revlimid Celgene Small molecule Multiple myeloma
Avastin Roche Antibody (monoclonal) Colorectal cancer; breast cancer;
non–small cell lung cancer; ovarian,
brain, and cervical cancer
Herceptin Roche Antibody (monoclonal) Breast cancer, gastric cancer
Enbrel Amgen Recombinant protein Rheumatoid arthritis, psoriasis
Prevnar 13 Pfizer Vaccine Pneumococcal (Streptococcus
Pneumoniae) antibacterial vaccine
Lantus Sanofi Peptide Diabetes mellitus types 1 and 2
Nelasta Amgen Recombinant protein Anemia (neutropenia/leukopenia)

5 The footballer Messi was treated with growth hormone, because he was only 1m60. But this raised ethical
issues: was this really necessary for Messi? 8 Recently, insulin can be extracted from pigs.
6 Recently insulin can be extracted from pigs
7 TPA was discovered by Désiré Collen, and this product was used to dissolve blood clots (bloedklonters)

causing strokes. He constituted the TromboGenics to invest in new research.

8

,  Most drugs are developed to combat diseases affecting humans

 Use genetically modified cultured cells to make protein of interest

In cell culture, cells are grown in dishes or flasks within liquid culture media designed to provide the
nutrients necessary for cell growth. Large culture containers, called fermenters or bioreactors, are used
to mass produce cells containing the DNA of interest.

 Gene therapy approaches: in the near future genes may be routinely introduced into human cells as
gene therapy approaches are employed to treat and cure human disease conditions. Genetics and
tissue engineering may lead to the ability to grow organs for transplantation that would only rarely
be rejected by their recipients. New biotechnology products from marine organisms are being used
to treat cancers, strokes, and arthritis. Specialized proteins, needed in quantity, will continue to come
from additional gene transfer to animals (like ATryn from transgenic goats; see CH7).

 Ethics and biotechnology: biotechnology is a controversial science that presents many ethical
dilemmas.

 Are GMO’s Good or Bad? https://www.youtube.com/watch?v=7TmcXYp8xu4

9

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