Transcript Biotechnology Lab

Genetic Engineering Lab
Bio 101A
April 10, 2008
• Describe your results from the PCR lab. Was
your sample GMO? How do you know?
• Describe differences between prokaryotes and
eukaryotes.
Brief Overview of Lab Objectives
1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)
2. Cut DNA into specific pieces using special enzymes
(restriction enzymes- BamHI; HindIII)
3. Measure size of pieces cut by enzymes (gel
electrophoresis)
4. Glue pieces together using other enzymes (DNA
ligase)
5. Take glued pieces and put them into another
bacterium (plasmid transformation of E. coli)
6. Separate bacteria with plasmid from those without
(antibiotic selection)
Today’s Objectives
1. Obtain Bacterial DNA (plasmids-pAMP and
pKAN)
2. Cut DNA into specific pieces using special
enzymes (restriction enzymes- BamHI;
HindIII)
Schedule
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9am- 910: Book check
910-915: Review questions
915-935: Introduction to lab
935-10am: Set up restriction digest/cleanup
10am-11am: restriction digest
10am-11am: Chi square discussion/practice
11am- refrigerate samples
Lab Concepts in Detail
Two Types of DNA in E. coli
Chromosomal DNA – necessary for cell survival; circular, double-stranded
Plasmid DNA – extrachromosomal DNA (“bonus material”) useful for experimental
manipulation; circular, double-stranded
Plasmids contain nonessential (but important) genes
β-lactamase can destroy penicillin
and other β-lactam antibiotics
Kanamycin interferes with
Ribosomes
• 30S ribosomal subunit
is affected
• Causes frameshift in
translation
• Toxic to humans
Plasmids can be cut with restriction enzymes
Enzymes homodimerize to make symmetrical cuts
BamHI
CGGATCCA
GCCTAGGT
CG
GCCTAG
GATCCA
GT
“sticky ends”
Restriction Enzymes cut very specific
sequences of DNA
Bacterium
Gene inserted into
plasmid
Plasmid
DNA
manipulation is at
the heart
of biotechnology
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Gene of
interest
Recombinant
DNA (plasmid)
DNA of
chromosome
Plasmid put into
bacterial cell
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
λ Phage is a temperate
bacteriophage
• Infects E. coli
• Genome is 46,000bp
long
• dsDNA
• Sequence is known
• HindIII-digested
genome is used as a
molecular marker
(ladder)
λ Phage digest is a common marker
• HindIII digest of phage
genome always yields
the same bands
• Draw pictures of what you expect in the
microfuge tubes from last week. Include as
much detail as possible. What did the
plasmids look like? What do they look like
now? What else is in the tube?
Objective(s) of the lab
• 1. Digest pAMP and pKAN with BamHI and
HindIII restriction enzymes
• 2. Determine size of plasmids using
electrophoresis
• 3. Create double antibiotic resistant plasmid
using DNA ligase
• 4. Transform E. coli with new plasmid
• 5. Select for transformants using antibiotic
media plates
Today’s Objectives
1. Ligate pAMP fragment to pKAN fragment
2. Determine fragment sizes using
electrophoresis with HindIII λ phage digest
Schedule
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8:10-8:20 Lecture spiel
8:20-8:55 Denature/Pour gel
9-10:15 Set up/Run gel
9:30-10:15 Discuss last quiz/Drosophila/Chisquare
• 10:15-10:30 Visualize gel
• 10:30-10:50 Create semilog graphs of
digest/determine fragment sizes
• 10:50-11 clean up
BamHI
BamHI
pKAN
pAMP
HindIII
ampR
Ori
BamHI
kanR
ampR
HindIII
Restriction digest
Ori
Ori
Ori
BamHI
HindIII
HindIII
BamHI
HindIII
kanR
Ligation
)
BamHI
kanR
ampR
HindIII
Ori
T4 is a Lytic bacteriophage
• Why might a lytic bacteriophage need DNA ligase?
Undigested plasmids are often
supercoiled
• Supercoiling- increased
or decreased number of
twists/bp
• Can be caused by
topoisomerases (type I
and type II)
Topoisomerases can cut DNA once
or twice
• Either way can increase
or decrease supercoiling
• Dimers can be made or
removed by
topoisomerases
Supercoiled, relaxed and linear
DNA do not run equally
• Why is supercoiled
faster than linear?
• Why are dimers slower
than monomers?
Week 3: Transforming Bacteria
1.
2.
3.
4.
Review Questions
What is our objective for the lab?
What was accomplished for this task last week?
How did what was done last week further our
objectives for the lab?
Define the following:
a. Plasmid
b. Ligase
c. Restriction Enzyme
Week 3: Transforming bacteria
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Outline for today
15 min. introduction
Transformation protocol (45 min.)
Incubation (60 min)
During incubation- Outline of selection (20 min);
completion of worksheet (due Wed. noon)
Plating- 30 min.
Predict plating results- (10 min)
Bacterial Transformation
• We will use chemically competent E. coli cells
• CaCl2, ice incubation, and heat shock facilitate the
process
Procedure
• Add 200 uL of
competent bacteria to
+LIG vial
• Add 200 uL bacteria to
any controls
• Gently mix
• Incubate on ice for 20
min
Procedure, cont.
• Heat shock for 90 sec.
• Place back in ice for
min. 60 sec.
• Add 800uL sterile LB to
tube
• Incubate on shaker for
60 min.
Sterile technique reminder
• Bacteria are ubiquitous
• Flame kills bacteria
• Any contaminants will
compete with our
bacteria of interest
Micropipettors Review
Are fragile
Expensive
Precise
They depend on correct usage for accuracy
Competent cells
• Transformation rate in
normal cells is low
• Transformation rate in
competent cells is
higher
• We use CaCl2 to make
cells chemically
competent
How can we determine if our
transformation was a success?
Selecting for transformants
• Antibiotic-infused agar
media permits only
resistant bacteria to
grow
• Our plasmids confer
specific antibiotic
resistance