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Topic
Cloning and analyzing oxalate degrading enzymes to see if
they dissolve kidney stones with Dr. VanWert
Topic
Cloning and analyzing oxalate degrading enzymes to see if
they dissolve kidney stones with Dr. VanWert
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
3. Pick some enzymes to clone and express
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
3. Pick some enzymes to clone and express
• Design primers
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
3. Pick some enzymes to clone and express
• Design primers
• Extract DNA (or RNA) from suitable source
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
3. Pick some enzymes to clone and express
• Design primers
• Extract DNA (or RNA) from suitable source
• Clone into PET plasmid
Game plan
1. Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
2. Learn more about candidate enzymes
3. Pick some enzymes to clone and express
• Design primers
• Extract DNA (or RNA) from suitable source
• Clone into PET plasmid
• Express and analyze
1.
2.
3.
4.
5.
Game plan
Learn more about kidney stones, conditions in kidneys,
or whether reducing oxalate elsewhere might work to
identify which enzymes might work best
Learn more about candidate enzymes
Pick some enzymes to clone and express
Design some experiments
See where they lead us
Grading Proposal
1. 5 assignments @ 5 points each
2. Draft of intermediate report: 5 points
3. Intermediate report: 10 points
4. Final presentation: 10 points
5. Poster: 10 points
6. Draft of final report 10 points
7. Final report: 30 points
Assignment 1
Pick an enzyme/organism combination
• Try to convince the group in 5-10 minutes why yours
is best
Genome Projects
Studying structure & function of genomes
C-value paradox
Size of genomes varies widely: no correlation with species
complexity
Cot curves
eucaryotes show 3 step curves
Step 1 renatures rapidly: “highly repetitive”
Step 2 is intermediate: “moderately repetitive”
Step 3 is ”unique"
Molecular cloning
To identify the types of DNA sequences found within each
class they must be cloned
Why?
To obtain enough copies of a specific sequence to work
with!
typical genes are 1,000 bp cf haploid human genome is
3,000,000,000 bp
average gene is < 1/1,000,000 of total genome
Molecular cloning
How?
1) introduce DNA sequence into a vector
• Cut both DNA & vector with restriction enzymes,
anneal & join with DNA ligase
• create a recombinant DNA molecule
Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
3) identify hosts which have
taken up your recombinant
molecules
Molecular cloning
How?
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
3) identify hosts which have
taken up your recombinant
molecules
4) Extract DNA
Vectors
Problem: most DNA will not be propagated in a new host
1) lacks origin of replication that functions in that host
Vectors
Problem: most DNA will not be propagated in a new host
1) lacks origin of replication that functions in that host
2) lacks reason for host to keep it
DNA is expensive!
synthesis consumes 2 ATP/base
stores one ATP/base
Vectors
Solution: insert DNA into a vector
General requirements:
1) origin of replication
2) selectable marker
3) cloning site: region
where foreign DNA
can be inserted
Vectors
1) plasmids: circular pieces of”extrachromosomal” DNA
propagated inside host
•origin of replication
•selectable marker
(usually a drug
resistance gene)
Multiple cloning site
• Upper limit:
~10,000 b.p. inserts
Transform into host
Vectors
1) Plasmids
2) Viruses
• must have a
dispensable region
Viral Vectors
find viruses with a dispensable region
Replace with new DNA
Package recombinant genome into capsid
Infect host
Viral Vectors
1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host
(cf < 0.01% for transformation)
Viral Vectors
1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host
(cf < 0.01% for transformation)
2) viruses are very good at forcing hosts to replicate them
may not need a selectable marker
Viral Vectors
1) viruses are very good at infecting new hosts
transfect up to 50% of recombinant molecules into host
(cf < 0.01% for transformation)
2) viruses are very good at forcing hosts to replicate them
may not need a selectable marker
Disadvantage
Viruses are much harder to work with than plasmids
Vectors
Viruses
• Lambda: can dispense with 20 kb needed for lysogeny