Welcome to BIOL301

Molecular and Cell Biology Laboratory

Hugo Zheng

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F2 plants

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Welcome to BIOL301
Molecular and Cell Biology Laboratory
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)
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Welcome to BIOL301
Molecular and Cell Biology Laboratory
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)
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Welcome to BIOL301
Molecular and Cell Biology Laboratory
11 1-hour lectures
11 6-hour labs
Experiments
EDMs experimental design/analysis modules

Lab coordinators: Anne-Marie Sdicu

Evaluation
60% labs (Worksheet + Quiz)
10% midterm + 30% final problem solving based!
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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 wellcontrolled experiments to study a biological problem,
and to learn how to make a valid conclusion based on
the results

Functions of genes, proteins, enzymes,

lipids, carbohydrates, hormones

What

When

How?
(mechanism)

knowledge

Where

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

Basic Methods of Inquire in Biology

root
a gene encodes a protein, which is a kinase that modifies protein Y that acts in intracellular signaling required for development

Basic Methods of Inquire in Biology

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mutant screen and
mappingbased cloning

3
bioinformatics

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expressed in root
meristem
5
Localized to PM

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micro-array and/or
2D-PAGE analysis

root
a gene encodes a protein, which is a kinase that modifies protein Y that acts in intracellular signaling required for development
regulate gene

mutation
1

- BiFC or FRET 6
- 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

7 expression,

protein
accumulation and
modification

phenotype
short roots

Focus of BIOL301 lectures

1. Gene identification: forward/reverse genetics in model species
2. Molecular cloning of a gene: Map-based cloning and
confirmation 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 complex
8. Protein characterization: SDS-PAGE and 2D-PAGE for posttranslational modification
9. Protein characterization: Western blot

Gene identification
genes are normally identified in a genetic model organism
Representative model to determine how things may work in
larger 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 & genomic
resources available

Saccharomyces cerevisiae has been used as a

minimal model eukaryote

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

Arabidopsis thaliana is chosen as a model

plant species

~20cm

1. Cell biology
2. Physiology
3. Gene regulation
4. Tissue patterning
5. Plant immunity

1cm

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

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

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 in
that process
wild type

mutant

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
T-DNA

insertional mutagens

Gene

mutagenize genes by sticking a large chunk of DNA in the

middle of it or its regulatory sequences
e.g. transposable elements, T-DNA in plant

You are interested in how plants control their

growth (comprised of many processes)

Arabidopsis thaliana

How do you find out which gene controls what process?

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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

Lab #1
Part One: Introduction to lab
satety intro: how to handle yourself, equipment & chemicals in the
lab
Techniques/skills required for mutant isolation in Arabidopsis

Part Two: Using learned techniques/skills

mutant identification shorty mutants of Arabidopsis
quick plant genomic DNA extraction (will be used for PCR in Lab #2)
wild type

shorty

wild type

shorty

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Goals of Lab #1-#5

wild type

shorty

In the Lab #1, you will identify an Arabidopsis mutant with a

growth defect, which you named shorty: 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.
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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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