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MODERN BIOTECHNOLOGY: What am I supposed to know…
MBB1 (1stAY2013-‐2014) MMP Balolong
LECTURE CONTENT: PART 1
Ê DNA: the genetic material (ONE GENE-‐ONE PROTEIN) Ê Prokaryotic system Ê Eukaryotic system
Ê INHERITANCE: the information connection, how genes are passed and their consequence Ê Prokaryotic system Ê Eukaryotic system
LECTURE CONTENT: PART 2
Ê MODERN BIOTECHNOLOGY Ê The GMO concept Ê How to design/develop a GMO Ê Tool/Methods: Gene Cloning, Genetic Engineering, Recombinant DNA technology
Ê Equipment and Facilities for GMO development
Ê Skills and Credentials
LECTURE CONTENT: PART 3
Ê MODERN BIOTECHNOLOGY : advances in gene-‐based applications Ê The GMO in Health and Medicine Ê The GMO in Industry Ê The GMO in Food and Agriculture Ê The GMO in Environment
TARGET ACTIVITIES
Ê WHO KILLED THE VICTIM? And WHO’S YOUR PAPA? (An activity on Forensics)
Ê EXPOSURE TRIP (July 24, 2013) Ê BIOTECH, UPLB Ê IRRI Ê IPB Ê PHIL CARABAO CENTER
www.crystalgraphics.com
THE 3 DOMAINS OF LIFE
bio1151b.nicerweb.net
gcps.desire2learn.com
BACTERIA & ARCHAEA
https://ib-‐biology2010-‐12.wikispaces.com
PROKARYOTES VERSUS EUKARYOTES
CHROMOSOME & DNA
DNA & THE CENTRAL DOGMA
www.labgrab.com
DNA: THE GENETIC MATERIAL
Ê WHY DNA? Ê The Griffith Experiment
Ê Concept of a transforming principle
Ê The Avery, McLeod and McCarty Experiment Ê That DNA is the transforming principle
Ê The Hershey and Chase Experiment Ê Transduction (via phage)
GRIFFITH’S EXPERIMENT:BACTERIAL TRANSFORMATION
AVERY, MCLEOD & MCCARTY: DNA AS TRANSFORMING PRINCIPLE
ap-‐biology-‐lab.wikispaces.com
HERSHEY & CHASE EXPERIMENT: TRANSDUCTION
SO HOW IS DNA, THE GENETIC MATERIAL, USED FOR BIOTECHNOLOGY?
THE SHIFT: TRADITIONAL TO MODERN
Ê Nature’s variation Ê Color, Size
Ê Traditional breeding can give us additional colors and sizes
Ê Modern Biotechnology can give you MORE… Ê Resistance to environmental
stress Ê Taste Ê Nutrition
kendralindell.wordpress.com
WHAT WE CAN DO WITH BIOTECHNOLOGY…
nakedmaninthetree.wordpress.com
www.ent.iastate.edu
www.flickr.com
www.ishs.org
GROUP WORK…
Ê One GMO per group
Ê Research on: Ê What problem want to be addressed by the GMO
Ê How was it developed
Ê What were the problems encountered
Ê How were they resolved (problems)?
Ê ISSUES….
Ê Open Discussion next meeting…
LAST MEETING… DNA and its importance in BIOTECHNOLOGY
1. Golden Rice; 2. BT Corn; 3. Edible Vaccine in Potato;
4. GMO Soya; 5. Recombinant Hepatitis B vaccine
RECALL…IN MODERN BIOTECHNOLOGY…WE CAN CONTROL THE VARIATION
Ê NOTE:
Ê Allelic differences at genes control a specific trait Ê GENE: a piece of DNA that controls the expression of a trait
Ê ALLELE: alterative form of the gene (they can segregate to form the variation naturally)
DNA (gene)
RNA
Protein Trait (or phenotype)
Transcription
Translation
Plant height
Seed shape
Traditional Breeding Biotechnology a. Gene manipulation b. Gene Introduction
Ê GMO or Genetically-‐Modified Organisms
Ê Organisms with ARTIFICIALLY-‐ALTERED DNA Ê Foreign gene is inserted from another species:
(enables GMO to express the trait coded by the new gene) = TRANSGENICS
Ê An existing gene is altered: to make it express at a higher level or in a different way = FOR GENE THERAPY
Ê Gene is deleted or deactivated: to prevent the expression of a trait (e.g. delayed ripening)
THE GOLDEN RICE
Ê WHY: to address Vitamin A deficiency problem Ê Linked to blindness Ê Influences Diarrhea and Measles Ê >100M children affected
Ê Vitamin Delivery as PILLS = most countries no access
Ê Alternative = vitamin into CROPS
www.goldenrice.org
β-‐Carotene Pathway in Plants
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
β -‐carotene (vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-‐beta-‐cyclase
ξ-‐carotene desaturase
Problem: Rice lacks
these enzymes
Normal Vitamin A “Deficient”
Rice
(Isopentenyl diphosphate)
The Golden Rice Solution
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
β -‐carotene (vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-‐beta-‐cyclase
ξ-‐carotene desaturase
Daffodil gene
Single bacterial gene; performs both functions
Daffodil gene
β-‐Carotene Pathway Genes Added
Vitamin A Pathway
is complete and functional
Golden Rice
THOUGH METABOLIC PATHWAYS ARE COMPLEX……THEY ARE INTERRELATED
Understanding pathways is critical to developing new
products
Modifying Pathway Components Can Produce New Products
Modified Lipids = New Industrial Oils
Turn On Vitamin Genes = Relieve Deficiency
Increase amino acids = Improved Nutrition
DEVELOPING OUR GMOs: Tools that you can USE
Ê Genetic Manipulation or Genetic Engineering Ê GOAL: introduce, alter, delete
Ê Cloning and Cell Culture Ê What to manipulate: Genes, Cells, Tissue
Ê GENOMICS, PROTEOMICS, METABOLOMICS
www.earthtimes.org
METHODS IN GENETIC ENGINEERING
Ê GENE ISOLATION
Ê TRANSFORMATION
Ê SELECTION
Ê REGENERATION
Ê CONFIRMATION OF EXPRESSION
EXAMPLE: INSULIN BY BIOTECHNOLOGY
muirbiology.wordpress.com
EXAMPLE: BT CORN
www.scq.ubc.ca
www.scq.ubc.ca
GENE ISOLATION
globalgenes.org
www.bluesci.org
www.cvillepodcast.com
hms.harvard.edu
THE PRINCIPLE TO ISOLATE THE GENE: Complementary Genetics 1. Protein sequence is related to gene sequence
NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-
ATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G
2. The genetic code information is used to design PCR primers
Forward primer: 5ʼ’-‐ATGGAT/CGCN-‐3ʼ’ Reverse primer: 5ʼ’-‐T/CTTNC/GT/ACCA-‐3ʼ’
Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence
THE GENETIC CODE
plato.stanford.edu
3. Use PCR to amplify gene fragment a. template DNA is melted (94C)
3ʼ’ 5ʼ’ 5ʼ’ 3ʼ’
3ʼ’ 5ʼ’ 5ʼ’ 3ʼ’
b. primers anneal to complementary site in melted DNA (55C)
3ʼ’ 5ʼ’ 5ʼ’ 3ʼ’
3ʼ’ 5ʼ’ 5ʼ’ 3ʼ’
c. two copies of the template DNA made (72C)
Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated
PCR Animation
Human clone library
Clones transferred to filter
PCR fragment probe added to filter
Hot-‐spots are human gene of interest
4. Gene fragment used to screen library
HOW WERE YOUR GENES ISOLATED?
Ê Golden Rice
Ê BT Corn
Ê Edible Vaccine in Potato
Ê GMO Soya
Ê Recombinant Hepatitis B vaccine
www.designingdigitally.com