Presentation done in MS university Baroda. "How to identify an yeast strain using molecular bio techniques?"...

1. IDENTIFICATION OF YEAST BY USING MOLECULAR APPROACH BY SINI P.K. REG.NO. 60415 St.Berchmans’ college Mahatma Gandhi University Kottayam,Kerala.

2. INTRODUCTION

3. INTRODUCTION Yeast strain isolated from gut of termite Named as Ter 10/T 10 Utilize pentose sugar (xylose) for alcohol production Xylase enzyme present Industries now using hexose sugar utilizing yeast Pentose sugar easily available and cheap Pentose sugar utilizing yeast is more economical

4. OBJECTIVES

5. Amplification of 26S rDNA in yeast genome Cloning of amplified 26S rDNA of yeast genome in KS plasmid OBJECTIVES

6. 26sr DNA of yeast genome Nuclear encoded ribosomal DNA of larger subunit Sequence Unique in an yeast strain Sequencing of 26S rDNA and comparing with data base for identification of yeast strain D1/D2 domains are unique(600–650 nucleotides) primers used are ITS1 and NL4. Cloning of this gene in plasmid makes sequencing and invivo and invitro studies technically more easier

7. MATERIALS AND METHODS

10. MATERIALS AND METHODS Yeast strain (T- 10) grown in YEPD broth Genomic DNA extraction of T-10 Amplification of 26S rDNA by PCR End filling of amplified PCR product Competent cell preparation using cacl2

11. MATERIALS AND METHODS Digestion of KS plasmid at EcoRv site Cloning of 26srDNA into EcoRv site of KS Transformation of KS plasmid to DH5alpha competent cell Selection of recombinants by Blue-White screening

12. MATERIALS AND METHODS Confirmation of clone. Preservation of clone

13. RESULTS & DISCUSSION

14. RESULTS & DISCUSSION Lane 1 – Genomic DNA from yeast Lane 2 – Genomic DNA from yeast Lane 3 – Genomic DNA from yeast RESULT 1: Isolation of genomic DNA from yeast (Ter 10)

17. Lane 2: KS plasmid digested with EcoRV

19. SUMMARY

20. summary 26s rDNA gene was amplified using the primer pairs ITS1 and NL4. Amplicon was then end filled with the enzyme T4 DNA polymerase and ligated in pBS KS+. Primary selection of putative transformed clones was performed on the basis of blue-white selection. 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. rDNA gene can be further sequenced and analyzed for its identification at the species level.

21. FUTURE PROSPECTS

22. Future prospects Identification of T 10 by sequencing of 26S rDNA Studies on pentose utilizing ability for ethanol production in industrial scale Optimisation of process parameters

23. “Thank you”