Strain Identification of yeast cell using Molecular Biology techniques

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"How to identify an yeast strain using molecular bio techniques?"...

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Strain Identification of yeast cell using Molecular Biology techniques

  1. 1. IDENTIFICATION OF YEAST BY USING MOLECULAR APPROACH<br />BY<br />SINI P.K.<br />REG.NO. 60415<br />St.Berchmans’ college<br />Mahatma Gandhi University<br />Kottayam,Kerala.<br />
  2. 2. INTRODUCTION<br />
  3. 3. INTRODUCTION<br />Yeast strain isolated from gut of termite<br />Named as Ter 10/T 10<br />Utilize pentose sugar (xylose) for alcohol production<br />Xylase enzyme present<br />Industries now using hexose sugar utilizing yeast<br />Pentose sugar easily available and cheap<br />Pentose sugar utilizing yeast is more economical<br />
  4. 4. OBJECTIVES<br />
  5. 5. Amplification of 26S rDNA in yeast genome<br />Cloning of amplified 26S rDNA of yeast genome in KS plasmid <br />OBJECTIVES<br />
  6. 6. 26sr DNA of yeast genome <br />Nuclear encoded ribosomal DNA of larger subunit<br />Sequence Unique in an yeast strain<br />Sequencing of 26S rDNA and comparing with data base for identification of yeast strain<br />D1/D2 domains are unique(600–650 nucleotides) <br />primers used are ITS1 and NL4.<br />Cloning of this gene in plasmid makes sequencing and invivo and invitro studies technically more easier<br />
  7. 7. MATERIALS AND METHODS<br />
  8. 8. MATERIALS AND METHODS<br />1. Bacterial strain<br /><ul><li> Escherichia coli DH5</li></ul>2. Growth medium for E. coli DH5<br /><ul><li> The E. coli DH5 was grown in Luria-Bertani (LB) medium. </li></ul>3. Yeast culture<br /><ul><li> Yeast isolated from termites was grown in YEPD medium</li></ul>4. Vector<br /><ul><li> Plasmid pBluescript KS+ was used as vector.
  9. 9. Size of plasmid is 2.9Kb</li></li></ul><li>Restriction map of pBS KS+<br />
  10. 10. MATERIALS AND METHODS<br />Yeast strain (T- 10) grown in YEPD broth<br /> Genomic DNA extraction of T-10<br /> Amplification of 26S rDNA by PCR<br /> End filling of amplified PCR product<br /> Competent cell preparation using cacl2<br />
  11. 11. MATERIALS AND METHODS<br />Digestion of KS plasmid at EcoRv site<br /> Cloning of 26srDNA into EcoRv site of KS<br /> Transformation of KS plasmid to DH5alpha<br /> competent cell<br /> Selection of recombinants by Blue-White<br /> screening<br />
  12. 12. MATERIALS AND METHODS<br />Confirmation of clone.<br /> Preservation of clone<br />
  13. 13. RESULTS & DISCUSSION<br />
  14. 14. RESULTS & DISCUSSION<br />Lane 1 – Genomic DNA from yeast <br />Lane 2 – Genomic DNA from yeast<br />Lane 3 – Genomic DNA from yeast<br />RESULT 1: Isolation of genomic DNA from yeast (Ter 10)<br />
  15. 15. RESULT 2:Amplification of 26s rDNA of yeast genome<br /><ul><li> Lane1- Amplicon size 1.3Kb
  16. 16. Lane 2- 1 kb ladder(1kb-10kb)</li></li></ul><li>RESULT 3: Restriction digestion of Blue script plasmid KS+ with EcoRV<br /><ul><li>Lane-1: Undigested KS plasmid
  17. 17. Lane 2: KS plasmid digested with EcoRV
  18. 18. Lane 3 : 1 kb ladder(1kb-10kb)</li></li></ul><li>RESULT 4: Cloning of 26srDNA into pBlueScript KS+<br />pBluescript KS+ was linearised using EcoRV. Then amplified 1.3kb fragment of Ter 10 was cloned into it through blunt end ligation.<br />Lane-1 : 100 bp ladder (100bp -1000bp)<br />Lane 2 : KS plasmid digested with BamHI and XhoI<br />Lane 3 : 1 kb ladder (1 kb – 10kb)<br />
  19. 19. SUMMARY<br />
  20. 20. summary<br /> 26s rDNA gene was amplified using the primer pairs ITS1 and NL4. <br />Amplicon was then end filled with the enzyme T4 DNA polymerase and ligated in pBS KS+.<br />Primary selection of putative transformed clones was performed on the basis of blue-white selection. <br /> Further confirmation of the clones was performed by Restriction Digestion with XhoI and BamHI. An expected band of 900bp-1.3Kb was obtained These transformed cells were preserved in glycerol stock and stored at -86 0 C.<br />rDNA gene can be further sequenced and analyzed for its identification at the species level.<br />
  21. 21. FUTURE PROSPECTS<br />
  22. 22. Future prospects<br />Identification of T 10 by sequencing of <br /> 26S rDNA<br />Studies on pentose utilizing ability for ethanol production in industrial scale<br />Optimisation of process parameters <br />
  23. 23. “Thank you”<br />

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