15/10/2021 1. biotechnology for everyone
TRANSCRIPT
15/10/2021
1
1. Biotechnology for everyone
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
From Traditional to Modern
LecturersMa. Cristina Francesca T. Dimaculangan, MScRoberta N. Garcia, PhD
Note that the training materials provided to you in any form is intended only for your use in connection with the this PSBMB online training course that you are participating in. They are not for distribution or sale. Permission should be obtained from PSBMB for any use other than for what it is intended.
Scope• Simple definition• Traditional biotechnology: Biotechnology
processes through the years• Modern biotechnology---genetic
engineering• Genome editing tools• Basic biotechnology laboratory techniques
Traditional biotechnology
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
15/10/2021
2
What is biotechnology?
Insert Texts Here
BIOTECHNOLOGY
Genetics Biochemistry
Engineering & computer science
medicine agriculture & forestry
environment industry
Other traditional disciplines
Mole-cularbiology
Involves many disciplines!
15/10/2021
3
Our Filipino ancestors practised biotechnology more than 400 years ago
From: History of the Philippine Islands VolsBy Antonio de Morga
• Their drink is a wine made from the tops of coconut and nipa palm… Drawing off the tuba, [235] they distil it, using …their own little furnaces and utensils, to a greater or less strength, and it becomes brandy. This is drunk throughout the islands. It is a wine of the clarity of water, but strong and dry. If it be used with moderation, it acts as a medicine for the stomach… protection against illnesses. with Spanish wine, it makes a mild liquor, and one very palatable and healthful.
Most traditional biotech products• Bread• Wine
• Tapuy• Tuba• Basi
• Vinegar • Soy sauce• Fish sauce• Miso• Natto• Tempeh• Kimchi
15/10/2021
4
More Biotech products through Biotransformation or bioconversion
•Conversion of substances to more useful forms by microorganisms• Formation of nutrients, amino acids,• Conversion of organic materials such as
plant or animal waste into usable products
Fermentation is a form of bioconversion
• Industry Applications
• Nutritionals and Pharmaceuticals
• Food and Beverage
• Plastic & Fibres
• Others
• s
Non-food bioconversion/fermentation products
World markets for fermentation revenue, by application;
US$ 47.4 Billion in 2016
https://www.credenceresearch.com/report/fermentation-chemicals-market
Plant breeding is biotechnology
➢3000 BC Peruvians started to improve potato to make them larger and frost tolerant.
15/10/2021
5
Bush tomato Australia
Different varieties of tomato
http://www.anfil.org.au/index.php?option=com_content&view=article&id=2&Itemid=104
wikipedia
Native corn
Modern corn
Hitchcock, A.S. (rev. A. Chase). 1950. Manual of the grasses of the United States. USDA Miscellaneous Publication No. 200. Washington, DC. 1950.
www.planet-diversity.org/
Native chickens from different parts of the world
Zhao et al, PAS Dec 2009 Modern breeds
Animal breeding is biotechnology
Fermentation
Tissue culture1960s
Biofertilizers1980s
Markers1990s
Recombinant DNA technology 2000s
Protein & DNA markers for disease diagnosis; crop varietal identity & diversity; marker-assisted breeding
Gene discovery; genetic engineering of crops for improved traits; protein engineering
BS, MS, PhD MBB Programs, UP Diliman (late1980s); MS, PhD UPLB (late 1990s); BS Ag Biotech UPLB (2010)
In the Philippines
Genomics
15/10/2021
6
Some homegrown biotech products
Tissue culture technologies•Embryo rescue of makapuno•Micropropagation of orchids
Dr. Emerita V. de Guzman
Dr. Helen Valmayor
Dr. Ramon C. Barba
Pioneered the induction of mango flowering using KNO3; Developed micropropagation techniques for banana and abaca.
Modern biotechnology
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
Scope of Biotechnology
Biofertilizers, biological nitrogen fixation, fermentation
Plant tissue culture
Cell culture
Recombinant diagnostics, DNA markers
Recombinant microorganisms
Genetic engineering of animals
Genetic engineering of plants
modern biotechnologyGENOMICS
Protein markers
Breeding
15/10/2021
7
Basis of genetic
engineering
❖What is a gene?
❖How is gene information converted to a trait?
❖What is recombinant DNA technology or genetic engineering
1866 Gregor Mendel discovered the basic laws of genetics---
❖Traits are inherited from one generation to another;
❖Elementen (genes) control the traits of organism;
❖there is a recombination of parental traits in offspring;
❖Some genes are dominant, some are recessive.
GOCarbohydratesFats
GROW Proteins
Glow Vitamins
CODE! Nucleic Acids
Molecules of Life
15/10/2021
8
What is a gene? DNA?
Gene
•a DNA
•codes for a trait
A Adenine
G Guanine
T Thymine
C Cytosine
A T
G C
DNAs are long chains of nucleotides with four different bases. The base is attached to the ribose sugar and the nucleotides connected by a phosphate group
A will always bind with T, and G with C; they are complementary to each other.
Thus allowing us to understand these basic genetic processes---
Why is the discovery of Watson and Crick very important?
• Based on the physical and chemical properties of DNA, Crick and Watson hypothesized a double-helix structure for DNA
• The double-helix structure explains
• How the DNA molecule could produce identical copies of itself (replication)
• How the information in the gene is expressed (transcription and translation)
15/10/2021
9
DNA DNA Since the nucleotide sequence of a strand is complementary to that of its partner strand, both strands will actually bear the same genetic information.
This process of DNA replication ensures that the resulting daughter DNA molecules are identical to the parental DNA helix.
Access Excellence@The National Health Musem
Information for trait is encoded in genes
• Some traits are simple; one gene for one trait• color, selected pest resistance traits
• Some traits are complex; many genes contribute to the formation of the trait• Yield, intelligence
gene
chromosomegenome
Totality of our traits
Information for a trait is encoded in the gene
15/10/2021
10
•The information in the gene is transcribed to a complementary single strand called messenger or mRNA; for mRNA, U (uracil) used instead of T (thymine)
•The mRNA travels from nucleus to cytoplasm and is translated to protein
Flow of genetic information
DNA (gene)
mRNA
protein
TRAIT
Flow of genetic information
Messenger RNA
In summary:
This basic knowledge is used in recombinant DNA technology or
genetic engineering
15/10/2021
11
• Why is it possible for a gene from an organism to be introduced and integrated in the genetic material of another organism, and also be expressed?
• For example, the genes for synthesizing Vitamin A from microorganism and flower can be introduced into the rice plant and be expressed in the rice grain!
Basis of Genetic Engineering or Recombinant DNA Technology
Cells of different organisms are similar
Their cellular constituents are also similar
They contain the same large and small molecules.
They follow the same chemical laws, physical laws and genetic laws.
Thus, a gene transferred to a new host organism can work and be expressed!
Genetic engineering or modern biotechnology allows the transfer of a specific gene for a
desirable trait
Recombinant DNA technology or genetic engineering---is a method that allows the combination of genes in a test tube to form a hybrid DNA.
•Design gene for specific trait
15/10/2021
12
1. Isolation of gene from source◦ Extraction of DNA
◦ Cloning of gene
2. Modify gene and prepare gene construct
3. Introduce to organism (transformation)
4. Improvement of transgenic plant (conventional breeding)
Steps in recombinant DNA technology or genetic engineering
promoter Gene XStructural gene
Terminator
original
New hybrid or rec DNA or gene construct
The gene and its parts
2. Modify gene and prepare gene construct
Insert modified gene in appropriate plasmid or vector
Let us compare conventional breeding with genetic engineering
15/10/2021
13
Conventional breeding
Genetic engineering
Gene of interest-1 page
Represents one book containing 1000 pages
Adapted from: Lemaux, Univ California, Publ. 8178, 2006
Our genome is like a library of books!
Genetic engineering of microorganisms
• Introduction of new trait in an organism
1
2
3
4
1- Isolate human insulin (HI) gene from human DNA
2- Insert HI gene in a vector
Production of human insulin by bacteria
3-transfection or transformation
Multiplication of plasmid; expression of gene
Genetic engineering
Gene
Agrobacterium
Particle bombardment
Gene transfer toplant tissue
Selection
Regeneration
Greenhouse selection/testing
Field testing
Variety
Gene
Vector
Marker gene Cloning sites
Gene construct
Tungsten bullets
shooting
infection
Step 1
Step 2Step 3
Step 4
1. Isolation of gene
– Extraction of DNA
– Cloning of gene
2. Modify gene and prepare gene construct
3. transformation
4. Improvement of transgenic plant
(conventional breeding)
Antibiotic resistance gene marker
+antibiotic
15/10/2021
14
Applications of recombinant DNA technology
• Production of important substances such as industrials and health products by microorganisms, plants and animals
• Improvement of traits of plants and animals• Using genetic engineering• Marker-assisted selection
• Design and production of designer foods
• Use in diagnostics• DNA Fingerprinting• DNA barcoding
• Use in basic research
• Basic tool in GENOMICS
When do we use genetic engineering in crop improvement?
• the trait to be introduced is not present in the germplasm of the crop
• the trait is very difficult to improve by conventional breeding methods
• it will take a very long time to introduce and/or improve such trait in the crop by conventional methods
Genome editing – gene editing
tools
15/10/2021
15
scope
• Definitions• Modern biotechnology
• Waves of biotechnology
• Gene editing tools
• Understanding gene editing principles
• Step-by-step procedure in gene editing
• Applications • Agriculture
• Health
First wave
Second wave
Third wave
Fourth wave
Modern Biotechnology Generations
Agronomic traitsBiotic or abiotic stresses
Quality traitsImproved nutrition, quality traits
Factories Industrials, pharmaceuticals
Renewable resources
Biofuels from cellulosic materials
These are the generations or waves of biotech processes or products
Most of the current products belong to the first wave.
Production of health products in microorganisms and mammalian cells
Fifth wave Use of gene editing tools
Modern biotechnology means the application of
Cartagena Protocol on Biosafety 2000
(a) in vitro nucleic acid techniques, including
recombinant DNA technology or direct injection of nucleic acid into cells or organelles
(b) Fusion of cells beyond the taxonomic family, that overcome natural physiological reproductive or recombination barriers and and that are not techniques used in traditional breeding and selection
•NEW: Gene editing tools like CRISPR-CAS 9, TALENs, Zn Finger Nucleases
15/10/2021
16
Recombinant DNA technology
Gene Target organism with new gene incorporated in its genome
New trait expressed
Can be higher or lower expressionor knockout!!
•Random insertion•Other parts of vector may be incorporated in target genome
NEW: Genome editing, gene editing tools
• Zinc finger nucleases (ZFN)
• TALENs (transcription activator-like effector nucleases)
• CRISPR-CAS
• Site of action is specific
• Can result in removal of single base or several bases or mutation to knock out gene or repair gene
• Can also insert a new gene for a new or improved trait
• Other parts of vector may be removed or not incorporated in genome
Genome Editing Tools(CRISPR Cas 9; Zn finger nucleases, TALENS, etc. )
• Enable editing parts of the genome by removing, adding or altering specific sections of the DNA sequence
• “Simplest, most versatile and precise method of genetic manipulation”
http://www.yourgenome.org/facts/what-is-crispr-cas9
15/10/2021
17
Nobel Prize in Chemistry 2020—for the development of a method of genome editing
Jennifer A. DoudnaUC Berkeley USA
Emmanuelle CharpentierMax Planck Unit for the Science of Pathogens, Berlin, Germany
Emmanuelle Charpentier and Jennifer Doudnadiscovered the CRISPR/Cas9 “genetic scissors” used to cut DNA.
Charpentier found the key tracerRNAmolecule that bacteria use to cut and disable viruses
collaborated with RNA expert Doudnato eventually ‘reprogram’ the scissors to cut any DNA molecule at a specific point, making the gene editing method viable.
CRISPR--- copying from nature
• Very old defense mechanism in bacteria
• In the 1980s, scientists observed a strange pattern of clusters of DNA repeats in bacteria, with unique sequences in between the repeats (clustered regularly interspaced short palindromic repeats or CRISPR)
• The unique sequences turned out to be bits of viruses that infect the bacteria which can recognize and defend against those viruses next time they attack.
Historical background
Then there is Cas!
• A set of enzymes called Cas (CRISPR-associated proteins) can precisely remove the DNA out of the invading viruses.
• The genes for the Cas are always near the CRISPR sequences.
• CRISPR is like a wanted gallery of invading viruses.
15/10/2021
18
CRISPR and Cas• The microbe can make the CRISPR-cas gun down
any invading virus! How?
• The spacer DNA sequences in the Gallery are copied into RNA molecules.
• These RNA molecules then bind with the Casenzymes and together drift through the cell.
• If they meet virus genetic material that matches and pairs with the CRISPR RNA, the Cas enzyme will then chop the DNA in two, preventing the virus from duplicating.
Gene editing
Invading virus
CRISPR repeats
CRISPR-cas will chop invading viral DNA/RNA
In bacteria
Virus parts
Just like antibodies in animals!
In Summary
15/10/2021
19
Steps in genome editing1. Gene construct—Cas9 plasmid, gRNA,
2. Delivery• Particle bombardment• Agrobacterium tumefaciens• Others (microinjection, liposome-mediated delivery)
3. Analysis of product• Sequencing• Studying phenotype etc
Biosafety Regulation
• To be discussed in a later lecture on biosafety regulation.
Some applications and products of gene editing-Agriculture
15/10/2021
20
Anti-browning White Button Mushrooms • Penn State University (2016)• Reduced formation of melanin,
improving shelf life of the mushroom
• CRISPR knock-out of one of six PPO (polyphenol oxidase) genes, reducing the enzyme’s activity.
• Waxy Corn (Zea mays)• DuPont Pioneer• Altered starch composition, 97%
vs 75%• CRISPR-deletion of waxy gene
Wx1
FAD2KO soybean• Hi Oleic acid, Low linoleic acid soybean oil• Intended effects: linoleic acid (5.04 vs 53.15%). Oleic acid
(77.13 vs 20.1 vs 17%); other changes are within range of scientific literature.
• TALENS-directed deletion within the FAD2-1A and FAD2-1B genes (enzymes for oleic acid and linoleic acid synthesis)
• Delivery thru Agrobacterium• Resulted in inactivation of the two genes
• Developed by the University of Tsukuba and Sanatech Seed.
• Fruit has 5 times the normal amount of GABA (gamma aminobutyric acid, an amino acid linked to lower blood pressure.) Dec 2020. Photo by Hiroshi Ezura of Tsukuba
University. https://asia.nikkei.com/Business/Science/Japan-approves-gene-edited-super-tomato.-But-will-anyone-eat-it
•Xanthomonas oryzae (Xoo) has transcriptor activator like effectors (TALEs).•TALEs have effector binding elements (EBE) in the promoter region of the OsSWEET genes.•OsSweet14 promoter gene has 4 EBEs.•Thru GEd, the 4EBEs were edited to obtain 4 different Super Basmati lines!
An earlier paper on similar topic. Oliva et al (2019) Nature Biotechnology 37: 1344-1350
Hornless bulls•Animals that do not have horns do not injure other animals, require less feeding trough space, are less dangerous to handle and transport than horned animals and have fewer aggressive behaviour. Their findings show that the rate of Cas9-mediated mutagenesis is not distinguishable from the background de novo mutation rate.
15/10/2021
21
In CHINA
Some of the GEd developments in China:•Virus resistant chicken. The Czech Academy of Sciences researchers developed chickens resistant to the common avian leukosis virus. Talks now of introducing this change into commercial breeds in China.•Tuberculosis resistant cows•Hardier pigs: Researchers at CAS used CRISPR to develop pigs that can withstand cold temperatures better and have leaner meat.•Fast growing pigs •Disease resistant pigs: resistant to classical swine fever and porcine reproductive and respiratory syndrome virus.
Some applications and products of gene editing-Health
Some applications of gene-editing tools in health
http://www.nature.com/news/crispr-gene-editing-tested-in-a-person-for-the-first-time-1.20988?WT.mc_id=TWT_NatureNews
http://www.foxnews.com/health/2016/11/08/stanford-uses-crispr-to-correct-sickle-cell-human-trials-planned.html
CRISPR edited cells with edited genes injected into a person with aggressive lung cancer
To treat sickle cells; single base mutation; tried in mice with some success
63
15/10/2021
22
https://www.sciencemag.org/news/2020/10/new-test-detects-coronavirus-just-5-minutes
Jennifer Doudna and Melanie Ott reported that with a single guide RNA they could detect as few as 100,000 viruses per microliter of solutionadding a second guide RNA, they detectd as few as 100 viruses per microliter.
• https://www.youtube.com/watch?v=_PU5bCflzEM Crispr video Crispr-Cas9 explained: the biggest revolution in gene editing J. Doudna
• https://www.youtube.com/watch?v=2pp17E4E-O8 Genome Editing with CRISPR-Cas9 Feng Zhang, McGovern Institute
Videos to Watch!
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
Basic Biotechnology Lab Skills
15/10/2021
23
• The micropipette is an important laboratory tool
• Accurate
• Transfer of small liquid volumes
• Virtual Lab Activity: https://bit.ly/3iGLZXp
Micropipette
• Manual Methods or Commercial Kits
• Sample Video: https://bit.ly/3uwMXKu
• Home-based Extraction: Banana
DNA Extraction
DNA Extraction Activity
Tecson – Mendoza, EM (2014)
15/10/2021
24
• Creates billions of DNA copies
• PCR Mechanism: https://bit.ly/3CVY2aK
• PCR Lab Method: https://bit.ly/3oi3D7k
Polymerase Chain Reaction (PCR)
• Uses an electric field
• Separate, purify and isolate nucleic acids
• Simulation Activity: https://bit.ly/2Y6E79P
• Lab video: https://bit.ly/3oK11iG
Agarose Gel Electrophoresis
• Protein separation based on mass
• Simulation activity: https://bit.ly/3imd1mK
• Lab Video: https://bit.ly/3kZSbv7
SDS - PAGE
15/10/2021
25
• Protein detection
• Check protein expression
• Laboratory simulation: https://bit.ly/3uti3SW
Western Blot
• Specific detection of RNA
• Analysis of RNA from sample
• Animation Video: https://bit.ly/3kUJHoO
Northern Blot
• Edward Southern, inventor
• Detects DNA
• Simulation videos https://bit.ly/3uwNoV8https://bit.ly/3F7fb3f
Southern Blot
15/10/2021
26
Enzymes Activity
Tecson – Mendoza, EM (2014)
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
1. Biotechnology for everyone
Simple definition
Traditional biotechnology: Biotechnology processes through the years
Modern biotechnology, genetic engineering
Genome editing tools
Basic biotechnology laboratory techniques
1. Biotechnology for everyone
National Academy of Science & Technology Philippines Copyright 2013; [email protected] Society of Biochemistry and Molecular Biology Copyright 2021; [email protected]
From Traditional to Modern
LecturersMa. Cristina Francesca T. Dimaculangan, MScRoberta N. Garcia, PhD
Note that the training materials provided to you in any form is intended only for your use in connection with the this PSBMB online training course that you are participating in. They are not for distribution or sale. Permission should be obtained from PSBMB for any use other than for what it is intended.