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8/9/2019 301Lect1 W2015
1/22
Controls F2 plants
CCLL LC 1 2 3
Welcome to BIOL301
Molecular and Cell Biology Laboratory
Hugo Zheng
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2
Lecture 1-5 and 12 (TBD)
Dr. Hugo Zheng
(focus on genes)
Lecture 6-10
Dr. Rodrigo Reyes-Ramothe(focus on proteins)
Lecture 11
Dr. Paul Harrison(bioinformatics)
Welcome to BIOL301
Molecular and Cell Biology Laboratory
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What you need: General Info Sheet + Syllabus (handout/myCourse)
Lab manual (McGill Bookstore)
Lab coat & safety goggles
Get a locker, no bags allowed in the lab
Reference texts Lodish (BIOL 200, 201 text book)
Griffiths (BIOL 202 text book)
Current Protocols Online eBooks (myCourse)
Context paper (myCourse)
3
Welcome to BIOL301
Molecular and Cell Biology Laboratory
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11 1-hour lectures
11 6-hour labs
ExperimentsEDMs experimental design/analysis modules
Lab coordinators: Anne-Marie Sdicu
Evaluation60% labs (Worksheet + Quiz)
10% midterm + 30% final problem solving based!
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Welcome to BIOL301
Molecular and Cell Biology Laboratory
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Purpose of BIOL 301
You will be expected to combine what you have
learned in your genetic and developmental or pathological classes and current molecular and cell
biology techniques learned in this class, to design well-
controlled experiments to study a biological problem,
and to learn how to make a valid conclusion based on
the results
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Functions of genes, proteins, enzymes,
lipids, carbohydrates, hormones
What When Where
How?
(mechanism)
knowledge
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Todays discovery requires an integrated approach
Hypothesis generation- bioinformatics
- 2-hybrid
- genetic screens
Hypothesis testing- RNAi,
- site mutations
- homologous recombination
- gene expression
- localization & dynamics
- protein interaction
& modification
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ageneencodes aprotein, which is a kinase that modifies protein Ythat acts in intracellular signalingrequired for developmentroot
Basic Methods of Inquire in Biology
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bioinformatics
ageneencodes aprotein, which is a kinase that modifies protein Ythat acts in intracellular signalingrequired for development
mutant screen and
mappingbased cloning
1
2
3
4
5
root
expressed in root
meristem
Localized to PM
mutation phenotype
short roots
Basic Methods of Inquire in Biology
micro-array and/or
2D-PAGE analysis
- BiFC or FRET
- Co-purification
- Protein modification:- extraction of proteins from wt
and mutant kinase background
- 2D-PAGE
- Western blot using anti-protein Y
to see possible changes
in between wt and mutant kinase
background
regulate gene
expression,
protein
accumulation and
modification
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7
8
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Focus of BIOL301 lectures
1. Gene identi ficat ion: forward/reverse genetics in model species
2. Molecular cloning of a gene: Map-based cloning andconfirmation of candidate gene
3. Study of gene expression: microarray, RT-PCR, Northern, in situ,
promoter-reporter gene fusion
4. Study of gene function: gene knockouts to analyze gene function,
RNA interference, over- and inappropriate gene expression
5. Protein localization and dynamics: GFP
6. Protein interaction: Methods for detection
7. Protein purification: tag-based protein purification to quantify
protein interaction in a complex8. Protein characterization: SDS-PAGE and 2D-PAGE for post-
translational modification
9. Protein characterization: Western blot
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Gene identification
genes are normally identified in a genetic model organism
Representative model to determine how things may work inlarger group of organisms focus on genetic models
Easy to work with
Grow fast
Lots of progeny
Small genome
Most have sequenced genomes
Easy to be manipulated to make transgenic individuals
Have international community of scientists working on same
system meetings, databases, stock centers, genetic & genomicresources available
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1. Protein secretion and membrane biogenesis
2. Function of the cytoskeleton
3. Gene regulation and chromosome structure
4. Control of cell cycle and cell division
1. single-celled eukaryotic organism
2. Undergo mitosis (90min) and meiosis
3. Grow rapidly in simple nutrient medium
4. Genome has 12.5mbp
5. ~6000 protein-coding genes
6. Easy genetic manipulation
Saccharomyces cerevisiae has been used as a
minimal model eukaryote
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Arabidopsis thaliana is chosen as a model
plant species
~20cm
1. Small in size, mature plants ~20cm
2. Can grow indoors in large number
3. Short life cycle, 8-10 weeks
4. Genome has 140mbp
5. ~20,000 protein-coding genes
6. Easy genetic manipulation
1cm
1. Cell biology
2. Physiology
3. Gene regulation
4. Tissue patterning
5. Plant immunity
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Forward versus reverse genetics in gene
identification
Forward genetics = classical genetics
going from phenotype to gene
widely used to identify genes acting in process of
interest
Reverse genetics
going from gene to function
requires a priory knowledge that makes you suspect a
gene is working in your process of interest
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Gene identification with forward genetics
Hunt mutant -- look for individuals with defects &/or
changes in your process of interest Conduct a genetic analysis for mutated gene(s):
recessive or dominant, single or multiple genes
Identify and clone gene(s) altered and/or involved inthat process
wild type mutant
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Mutagens used to increase frequency of
finding mutations & thus the number of genes
identified
chemical mutagens e.g. ethylmethane sulfonate (EMS)
point mutations -- affect proteins through truncation or loss of
key amino acids
radiation e.g. X-rays, fast neutrons leads to small or large deletions of DNA or even chromosome
breakage & rearrangements
insertional mutagens
mutagenize genes by sticking a large chunk of DNA in themiddle of it or its regulatory sequences
e.g. transposable elements, T-DNA in plant
Gene
T-DNA
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You are interested in how plants control their
growth (comprised of many processes)
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How do you find out which gene controls what process?
Arabidopsis thaliana
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How do you find out which switch controls what?
???
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Random mutating and isolating mutants with
phenotype of interest in the next generation
Random mutations
Individual with
a mutation in
a gene that is
needed for
proper growth
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Lab #1
Part One: Introduction to lab
satety intro: how to handle yourself, equipment & chemicals in thelab
Techniques/skills required for mutant isolation in Arabidopsis
Part Two: Using learned techniques/skills mutant identification shorty mutants ofArabidopsis
quick plant genomic DNA extraction (will be used for PCR in Lab #2)
20
wild type shorty wild type shorty
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Goals of Lab #1-#5
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In the Lab #1, you will identify an Arabidopsis mutant with a
growth defect, which you namedshorty: In the Lab #2-#5, you
will map (Lab #2), clone (Lab #3), confirm (Lab #4), and study
the expression pattern (Lab #5) of the gene.
wild type shorty
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Context Paper used throughout Lecture 2-3
Will be using this as an example
throughout Lect 2-3
Available on myCourse
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