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