Strain Identification of yeast cell using Molecular Biology techniques
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Transcript of Strain Identification of yeast cell using Molecular Biology techniques
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”