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CLONING AND PURIFICATION OF REPORTER CONSTRUCT PLASMID CONTAINING FULL
LENGTH PROMOTER REGION OF ONCOSTATIN-M
Presented by:-
DEBANJAN BANERJEEM.SC Microbiology
Registration no:-114-1121-0194-10VIJAYGARH JYOTISH RAY COLLEGE
Under the supervision of:-
Dr SUMTA SENGUPTA(Department of Molecular biology, Biophysics and
Bioinformatics)
UNIVERSITY OF CALCUTTA
EUKARYOTIC TRANSCRIPTIONAL REGULATION
Transcriptional regulation is said to be a control of gene expression by the cell at transcriptional level.
Transcription in the eukaryotic cells is regulated by the proteins which
bind to specific regulatory sequence and modulate the activity of RNA
polymerase. Sometimes, the packaging of the DNA into histones and modification
by methylation is involved into further complexity for gene regulation.
ONCOSTATIN-M
Oncostatin-M is a cytokine.
It belongs to interlukine-6(IL6) family It is molecular weight of 28 kd. Secreted by- Macrophages, T cells. Target cells- Tumor cells. Function- Inhibits the growth of the tumor cell.
PROMOTER OF ONCOSTATIN-M
IMPORTANT CORE ELEMENTSGC-rich element.STAT element.CRE region.
The prime focus is to isolate the promoter region1 kb upstream of ONCOSTATIN-M gene and clone it to a expression vector for LARGE SCALE PLASMID
ISOLATION for further assay.
AIM OF THIS PROJECT
WORK FLOW
Culture U937 cell line
Isolation of genomic DNA
Amplification of full length promoter region of osm gene from the genomic DNA
WORK DONE PREVIOUSLY
RESULT- FULL LENGTH PROMOTER REGION(PON12) AMPLIFICATIO FROM GENOMIC DNA
960 bp
1 2 3 4 5
Lane 1-1000 bp ladderLane 3-5-Amplified PON12 region
Ligation of the promoter region into TA cloning vector
Transformation into E.coli XL Blue cell
Colony selection and re-streaking for storage
Plasmid( pt-Pon12) isolation
RESULT-PLASMID ISOLATION OF pT-PON-12
1 2 3 4 5
Lane 1-PT-PON12 plasmidLane 3-Ptz plasmid without insertLane 5-PT-PON12 plasmid
Sub-cloning into pEGFP-1 vector with the help of directional
cloning by 2 different restriction enzymes (Eco RI and Bam HI)
Eco RI
Bam HI
Eco RI Bam HI
Pon 12
RESULT-Restriction digestion of pEGFP-1 and pT-Pon12 plasmids
1 2 3 4 5 6 7
960 bp
Lane 1-1000 bp ladderLane 3-Uncut PEGFP-1Lane 4-Digested PEGFP-1 by Eco RI, Bam HILane 6-PT-PON12 digested by Eco RI , Bam HILane7-PT-PON12 digested by Eco RI, Bam HI
Ligation and Transformation into E. coli XL Blue cells
Colony selection and re-streaking
Perform colony PCR for confirmation of clone
Selection of the positive colony
RESULT-Colony PCR from colony containing p-EGFP1-Pon12 plasmid with specific primer Pon1 and Pon2
1 2 3 4 5 6 7 8
960 bp
LANE 1,3,4,8-Colonies picked from transformed plateLANE 5- 1000 bp ladder
Inoculate this colony into LB +Amp media for large scale plasmid
culture
Grown overnight
Isolation of pEGFP1-Pon-12 plasmid by PEG and LiCl2 method
The isolated plasmid is used for the further assay
1 2
LANE1-pEGFP plasmid without insert
LANE2-pEGFP plasmid with insert
RESULT- ISOLATION OF pEGFP1-Pon12 PLASMID
CONCLUSION
Thus we have successfully cloned and isolated PEGFP1 vector containing full
length osm promoter region .This clone can be further used for transfection and
subsequent reporter assay in mammalian system. This will help us study the role
of osm promoter region in its efficient transcription.
ACKNOWLEDGEMENT Dr SUMITA SENGUPTA
Department of Molecular Biology, Biophysics BioinformaticsUNIVERSITY OF CALCUTTA
All my guides, specially,
SRIMOYEE MUKHERJEE
PROSENJIT DASHead of The Department, Microbiology
Vijaygarh Jyotish Ray College
All my respectable professors, department of MicrobiologyVijaygarh Jyotish Ray College
CALCUTTA UNIVERSITY CENTRAL INSTRUMENTAL FACILITY
Thank You
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