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Dictyostelium discoideum cloning putative gene & Life cycle

Dictyostelium discoideum cloning putative gene & Life cycle

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Bhupi dicty Presentation Transcript

  • 1. Summer Training PresentationsDepartment of Molecular and Human Genetics
  • 2. Understanding of the life cycle of Dictyostelium discoideum and cloning of putative gene Mentor: Dr. Shweta Saran Developmental Biology Lab SLS JNU, New Delhi.Presented By:Bhupender VermaM.Sc. (F) MHG BHU
  • 3. Introduction Dictyostelium discoideum are singlecellular organismsLive on decaying logs and feed onbacteriaDictyostelium discoideum follow asexualcycle-• when there is plenty of food available• Live solitary• Are haploid• Reproduce by binary fission Also called as “Social amoebae”During starvation organisms aggregateand migrating slug is formed thatculminates in a fruiting body to releasespores
  • 4. Figure: Scott F. Gilbert-Developmental Biology 8th edition
  • 5. Classification:Domain – EukaryaKingdom- AmoebozoaSuperphylum- ConosaPhylum- MycetozoaClass- DictyosteliaOrder- dictyosteliidaFamily- DictyosteliidaeGenus- DictyosteliumSpecies- D. discoideum
  • 6. Life cycle In lab Dicty are grown in HL5 liquid media at 22ºC. In nutrient rich conditions Dicty divide by binary fissionand remain solitary. For studying their development a concentrated solutionis transferred to the NNA plates. In the absence of nutrients, these myxomoebae jointogether to form moving streams of cells converging at acentral point. Different developmental stages that can be observed:• Tight aggregate• Migrating slug (pseudoplasmodium or grex) – slimysheath encased• Fruiting body
  • 7. Vegetative loose aggregate mound migratory slug Early culminant fruiting body
  • 8. Cloning of the putative gene:Strategy:• Design the primers• Gradient PCR for optimizing annealing conditions• Standard PCR for obtaining amplified insert• Digestion of vector and insert with HindIII and PstI• Liagtion of insert to the vector• Transform bacteria with recombinant vector• Isolate cloned plasmid and digest for checking if insert isdesirable
  • 9. Gradient PCR for optimizing annealing conditions:1 2 3 4 5 6 7 8 9 10 M 11 12 13 14 15 16 17 18 19 20 Design the primers • Gradient PCR 5 kb • Standard PCR 1.5 kb • Digestion with HindIII 500 bp and PstI • Liagtion • Bacterial 21 22 23 24 25 26 27 28 29 30 M 31 32 33 34 35 36 transformaton • Isolate cloned plasmid 5 kb and digest for checking 1.5 kb if insert is desirable 500 bp
  • 10. Standard PCR for obtaining amplified insert: Design the primers • Gradient PCR • Standard PCR • Digestion with HindIII 5 kb and PstI • Liagtion 1.5 kb • Bacterial transformaton 500 bp • Isolate cloned plasmid and digest for checking if insert is desirable
  • 11. pDrive cloning vector- Design the primers • Gradient PCR • Standard PCR • Digestion with HindIII and PstI • Liagtion • Bacterial transformaton • Isolate cloned plasmid and digest for checking if insert is desirable
  • 12. Digestion of vector and insert with HindIII and PstI Design the primers • Gradient PCR • Standard PCR • Digestion with HindIII and PstI 5 kb • Liagtion • Bacterial 1.5 kb transformaton • Isolate cloned plasmid and digest for checking 500 bp if insert is desirable
  • 13. Checking insert from fallout size: Design the primers 5 kb • Gradient PCR 1.5 kb • Standard PCR 500 bp • Digestion with HindIII and PstI • Liagtion • Bacterial transformaton 5 kb • Isolate cloned plasmid 1.5 kb and digest for checking 500 bp if insert is desirable
  • 14. Cloning two segments of a putative genein pDrive vector for making Knockout-1. Insert 1 – obtained from digestion (HindIII & NotI) of cloning vector as pop out2. pDrive vector – already had other insert so digest with (HindIII & NotI) & obtain vector-BSR3. Ligate insert to vector4. Transform bacteria5. Grow bacteria and isolate plasmid6. Digest plasmid for checking proper ligation7. Proceed with the colony that had proper insert8. PCR of insert 29. Digest insert 210.Ligation11.Transformation12.Isolate plasmid and check
  • 15. 1. Insert 1-as pop outDigestion with HindIII & NotI 2. pDrive -had other insert 3. Ligation of insert 1 4. Transform E.coli 5. Grow E.coli & isolate plasmid 6. Digest plasmid for check 7. Proceed with sample +BSR having proper insert 8. PCR of insert 2 9. Digest insert 2 10.Ligation 11.Transformation 12.Isolate plasmid and check
  • 16. 1. Insert 1-as pop outCheck after elution from 2. pDrive -had otheragarose gel: insert 3. Ligation of insert 1 4. Transform E.coli 5. Grow E.coli & isolate plasmid 6. Digest plasmid for 5 kb check 7. Proceed with sample 1.5 kb having proper insert 8. PCR of insert 2 500 bp 9. Digest insert 2 10.Ligation 11.Transformation 12.Isolate plasmid and check
  • 17. 1. Insert 1-as pop outFor checking again digest 2. pDrive -had otherwith HindII & NotI insert 3. Ligation of insert 1 4. Transform E.coli 5 kb 5. Grow E.coli & 1.5 kb isolate plasmid 500 bp 6. Digest plasmid for check 7. Proceed with sample having proper insert 8. PCR of insert 2 5 kb 9. Digest insert 2 1.5 kb 10.Ligation 500 bp 11.Transformation 12.Isolate plasmid and check
  • 18. 1. Insert 1-as pop outFor checking again digest 2. pDrive -had otherwith HindII & NotI insert 3. Ligation of insert 1 4. Transform E.coli 5 kb 5. Grow E.coli & 1.5 kb isolate plasmid 500 bp 6. Digest plasmid for check 7. Proceed with sample having proper insert 8. PCR of insert 2 5 kb 9. Digest insert 2 1.5 kb 10.Ligation 500 bp 11.Transformation 12.Isolate plasmid and check
  • 19. After insert1 what do we have? pDrive-Insert1-BSR
  • 20. 1. Insert 1-as pop outDigestion with HindIII & 2. pDrive -had other BamhI: insert 3. Ligation of insert 1 4. Transform E.coli 5. Grow E.coli & isolate plasmid 6. Digest plasmid for check 7. Proceed with sample 5 kb having proper insert 1.5 kb 8. PCR of insert 2 500 bp 9. Digestion of insert 2 10.Ligation 11.Transformation 12.Isolate plasmid and check
  • 21. 1. Insert 1-as pop out Result: 2. pDrive -had other insert 3. Ligation of insert 1 4. Transform E.coli 5. Grow E.coli & pDrive Insert1-BSR-Insert2 isolate plasmid 6. Digest plasmid for check 7. Proceed with sampleNow we are ready with a DNA having proper insert 8. PCR of insert 2segment that is “insert1-BSR- 9. Digestion of insert 2insert2” 10.LigationThis insert can be used for 11.Transformationknocking out this putative genes 12.Isolate plasmid andAnd my insert is still under study check
  • 22. References:RICARDO ESCALANTE and JUAN J. VICENTE- Dictyosteliumdiscoideum: a model system for differentiation and patterning(2000)L. Eichinger, J. A. Pachebat, G. Glockner- The genome of the socialamoeba Dictyostelium discoideum (2005)Developmental biology- Scott F. Gilbert- 8th edition.
  • 23. Thank you!