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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