Download 1 Biology 437 Fall 2015 Syllabus Biology 437: LABORATORY ON

Biology 437
Fall 2015
Biology 437: LABORATORY ON DNA MANIPULATION
Instructor: Professor Robert Kranz ( [email protected] )
Syllabus
TAs: Jared Andrews ([email protected] )
Nicole Fazio ( [email protected] )
Mary Mathyer ( [email protected] )
Sarah Santiago ( [email protected] )
Rebstock room 126: Wed & Fri 2:30-6pm; Thursday 4-5pm
Textbook: none ("Fermentas" catalog with technical information is used).
I.
You will be given the following handouts on the first day of class:
1. Student Questionnaire
2. Bio 437 course overview and goals
3. Class Syllabus (timing subject to change throughout semester)
4. Guidelines for keeping a Laboratory Notebook
5. Guide for the lab write-up
6. Laboratory Safety Rules
7. Handouts for this week’s lab (Lab I)
8. A lab notebook and 3-ring binder
II.
How is the class graded?
• Two in-term exams: 1st is 100 pts; 2nd is 50 pts
• Two Problem sets: 1st is 50 pts; 2nd is 25 pts
• Subjective 25 pts given after the course is over, decided by all
instructors (eg. missed or late labs, assignments, sloppy work)
• Lab Write-up (Paper): 100 pts
• Lab Notebook
50 points (collected twice, 25 pts each time)
Total
400 points
III.
Office Hours: (Dr. Kranz and/or at least two TAs will be at all labs and
lectures so these 8 hours per week will have ample time for discussion, or
alternatively, email Dr. Kranz)
IV.
Bring your lab notebook and binder to every period.
V.
Lectures will be interspersed into lab experiments where they fit.
VI.
Attendance is mandatory. Be on time because a short lecture outlining
the day’s procedure will take place at the start of some labs.
VII.
There is NO CLASS on Friday of Fall Break (Oct16). There are no
classes the week of Thanksgiving Break (Nov 25-29). Last day of classes
is Friday, Dec 4.
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Week (and
W,Th, Friday
Dates)
Lecture(s)
Experiments
Important info &
dates
1
Lect 1&2
Intro, DNA
Calibrate pipettes/sterile
technique
Cloning assignments will
be given.
Individual meeting about
cloning projects.
Primer design. Set-up E.
coli overnights to prep DNA
for cloning projects.
Preparation of DNA for
cloning projects.
Diagnostic digest of DNA &
DNA agarose gel.
Prepare oligonucleotides
(primers) for Quikchange
PCR reaction for sitedirected mutagenesis.
DpnI digest.
Transformation of reactions
into E. coli. Count colonies
from transformation.
Set-up overnights to prep
clone DNA.
Module 1 starts
(Aug 26,27,
28)
2
(Sept 2,3,4)
3
(Sept
9,10,11)
4
(Sept
16,17,18)
5
(Sept
23,24,25)
6
Lect 3&4
PCR,
molecular
cloning, sitedirected
mutagenesis
Lect 5&6
Biology
behind
module 1
projects
Prepare clone DNA.
Diagnostic digest and DNA
agarose gel.
Prepare sequencing
reaction.
Lect 7 DNA
sequencing
Analyze sequencing
reactions.
Set-up overnights to freeze
strains or if necessary,
troubleshoot cloning.
Lect 8
Homologous
recombinatio
n/knockouts
Trouble-shooting,
discussion of next
experiments.
Lect 9
Impact of
(Sept 30, Oct Genome
1, 2)
sequencing
Trouble-shooting,
discussion of next
experiments.
Wed: lecture,
pipetting,primer
design
Thur: Sterile
technique, start
E.coli cultures, finish
lecture
Fri: DNA minipreps
and analyses
Problem Set 1
handed out (due
week 6, Wed, Sept
30, 2:30pm)
Problem Set 1 due
week 6, Wed, Sept
30, 2:30pm)
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Lect 10 RNA
and RNA
methods
Trouble-shooting,
discussion of next
experiments. Protein work
on mutated genes
(proteins), if feasible
Lect 11 RNA
and RNA
methods
Trouble-shooting,
discussion of next
experiments. Protein work
if feasible
Exam 1 on Wed, Oct
14 (covers module 1
Continue protein work.
Module 2 starts
For Module 2, set up corn
seeds for roots vs shoots
analysis, RNA studies.
Module 1 will
continue as time
permits.
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Finish module 1.
(Oct
28,29,30)
Module 2: Design primers
for corn transgenes.
Practice tissue prep and
RNA purification Tissue
prep and RNA purification.
Problem Set 2
handed out (due
week 12, Wed, Nov
11, 2:30pm
(Oct 7,8,9)
8
(Oct 14,15,
no class on
Oct 16)
9
(Oct
21,22,23)
Lect 12
Biology
behind
Module 2
projects
11
Make cDNA.
(Nov 4,5,6)
Perform RT-PCR to
become familiar with the
technique.
12
Lect 13 Other
(Nov
11,12,13)
technologies in
nucleic acid
manipulations
13
(Nov
18,19,20)
(Dec 2,3,4)
Fall Break on Friday, Oct
16)
OCT 30 Halloween
pumpkin carving
Perform RT-PCR/QRTPCR. Analyze the results
Problem Set 2 due
Wed, Nov 11,
2:30pm
Trouble-shooting,
discussion of next
experiments.
Discuss which
project to write up
into paper.
Thanksgiving
(Nov 25-29)
14
and lectures 1 thru 9)
Thanksgiving
Lect 14
Summary of
Bio437
successes,
writing a
scientific
paper
Wrap-up final experiments
with Module 1&2
Exam 2 on Wed,
Dec 2—covers
module 2 and
lectures 9 thru 13
Clean up lab /DipN
Dots & pizza party
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Bio 437 (Fall, 2015): General Overview and two lab modules: from Prof Kranz
The magnificent boom in biotechnology since the 1970s is a direct result of the ability to manipulate and
measure nucleic acids. These advancements have revolutionized research in health and the
environment. This course is designed as a hands-on experience that will provide the student with an
understanding of how research on nucleic acids is performed and that also may be of use in the
biotechnology field. Module 1 is based on DNA technologies while module 2 provides hands-on research
in RNA methods.
Module 1) Cloning genes, directed mutagenesis, and sequence analysis:
Projects for Table A: Organisms use cytochrome c for respiration and photosynthesis. For example,
eukaryotes have mitochondria whereby cellular respiration via electron transport is used to synthesize
ATP and for other processes essential to life. All mitochondria possess cytochrome c (and cytochrome c1)
for this electron transport. All cytochrome c proteins have heme that is covalently attached to the protein
by two thioether bonds (at two cysteines in the protein, cys15-X-X-cys18-his19). This covalent attachment
requires the mitochondrial protein called holocytochrome c synthase (HCCS). Since the gene for HCCS
and other mitochondrial proteins are the targets of many human diseases and cytochrome c is also
needed for programmed cell death (apoptosis), it is important to understand how HCCS functions, and to
be able to control it. Very recently the Kranz lab was able to clone the human HCCS and the human
cytochrome c and show it is functional in Escherichia coli (ie the heme is attached by HCCS). This makes
it feasible to study HCCS and to potentially biosynthesize new types of cytochromes c. Bio437 students
will engineer into the human cytochrome c (gene) new amino acids (substitutions or deletions in codons)
that have never been made, using the genetically engineered E.coli to biosynthesize these novel
cytochrome c.
Projects for Tables B and C: Every organism on earth can use the 20 natural amino acids in
their coding sequences--- this is at the core of the “central dogma” of biology. A relatively new technology
has emerged whereby it is possible to “recode” a gene by genetic engineering, facilitating the use of nonstandard amino acids (NSAA). We can thus engineer anywhere in a protein this “new” NSAA by
genetically changing the codon and employing engineered protein synthesis machinery that will recognize
the new NSAA. For this class, we will genetically engineer (recode) genes that encode a cytochrome c
from bacteria (called cytochrome c4). This will allow for overproduction, purification and characterization of
cytochrome c proteins in Escherichia coli that now will incorporate the NSAA of our choice.
Main scientific questions: Those students constructing alterations in the human cyt c will test whether the
variants interact with HCCS and can be assembled with heme. For students “recoding” for NSAA, a
particular NSAA (eg. Benzoylphenylalanine) crosslinks to any other residue near it, so will properly
positioning this NSAA crosslink the cytochrome c to the cytochrome c synthetase that makes cytochrome
c? This would define where interaction is between the two proteins. Will these recoded cytochrome c’s
be functional? Will these engineered (recoded) cytochrome c proteins allow incorporation of fluorescent
NSAAs, to make entirely new chromophores? We hope to demonstrate how use of mass spectrometry
will prove that a NSAA has actually been incorporated into a cyt c, which has never been accomplished
before.
Training rationale: You will learn to design, clone, and analyze the sequence for the mutations. This
process is called genetic engineering and is critical for basic science, biotechnology, and biomedical
research. Through this project you will gain hands on skills in DNA manipulation that will be translatable
to a wide array of research fields. We will pursue overexpression in E.coli of the new cytochromes you
engineered and its analysis by spectroscopic and other techniques.
TAs: Sarah Santiago, Mary Mathyer, and Nicole Fazio
Module 2) RNA analyses: RT-PCR and q-PCR on mRNAs for gene expression. Students will carry
out projects using a eukaryote (i.e. the corn plant Zea mays). RNAseq, q-PCR, and microarrays are now
the gold standard technologies for comprehensive understanding of what genes are involved (i.e.
expressed) under certain conditions (or diseases). In previous years, Bio 437 students carried out
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microarray analyses and q-PCR of genes expressed in Arabidopsis (a weed) under nitrogen or phosphate
stress (ie. fertilizer-deficient plants). This year we will analyze genetically modified organisms (GMO corn)
that have engineered traits (“transgenes”) that farmers use to outcompete weeds and fight off insect pests.
These GMO corn have been transformed with genes (transgenes) from bacteria, called “round-up ready,
RR” or BT toxin genes. (The BT toxins (encoded by cry genes) kill insects, the RR gene is for resistance
to a herbicide called Round-up.)
The main scientific questions: address the levels and location of expression of BT (Cry) and RR genes in
GMO corn. Training rationale for this project: Learning the details of mRNA analysis, including tissue
preparation, RNA purification, cDNA synthesis, RT-PCR, q-PCR, global gene expression profiling. (eg.
many studies on global gene expression profiling of human cancer cells are unraveling what genes are
involved in the cancer state, and require these technologies for analyses).
TA: Jared Andrews
Other learning goals: Although we will not sequence or annotate an entire genome, we will emphasize
throughout the course how and why whole genome sequences have revolutionized biology and
biotechnology. A few lectures will cover other methods of biotechnology (nucleic acid manipulation) which
we will not use (eg. RNAi, CRISPRs, gene knock-outs, Chromatin immunoprecipitation, RNAseq).
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