IDENTIFICATION OF YEAST BY USING MOLECULAR

APPROACH

BYSINI P.K.

REG.NO. 60415St.Berchmans’ college

Mahatma Gandhi UniversityKottayam,Kerala.

INTRODUCTION

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

OBJECTIVES

Amplification of 26S rDNA in yeast genome

Cloning of amplified 26S rDNA of yeast genome in KS plasmid

OBJECTIVES

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

MATERIALS AND METHODS

MATERIALS AND METHODS

1. Bacterial strain Escherichia coli DH5

2. Growth medium for E. coli DH5 The E. coli DH5 was grown in Luria-Bertani

(LB) medium.

3. Yeast culture Yeast isolated from termites was grown in

YEPD medium

4. Vector Plasmid pBluescript KS+ was used as vector. Size of plasmid is 2.9Kb

Restriction map of pBS KS+

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

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

MATERIALS AND METHODS

Confirmation of clone.

Preservation of clone

RESULTS & DISCUSSION

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)

RESULT 2:Amplification of 26s rDNA of yeast genome

Lane1- Amplicon size 1.3Kb

Lane 2- 1 kb ladder(1kb-10kb)

RESULT 3: Restriction digestion of Blue

script plasmid KS+ with EcoRV

Lane-1: Undigested KS plasmid

Lane 2: KS plasmid digested with EcoRV

Lane 3 : 1 kb ladder(1kb-10kb)

RESULT 4: Cloning of 26srDNA into pBlueScript

KS+pBluescript KS+ was linearised using EcoRV. Then

amplified 1.3kb fragment of Ter 10 was cloned into it through blunt end ligation.

Lane-1 : 100 bp ladder (100bp -1000bp) Lane 2 : KS plasmid digested with BamHI and XhoI Lane 3 : 1 kb ladder (1 kb – 10kb)

SUMMARY

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.

FUTURE PROSPECTS

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

“Thank you”