SUMMER TRAINING REPORT

ON

Cloning of OsiSpo11-1 cDNA  in Plant Expression Vector and its Transformation in Agrabacterium tumefaciens for its Over-expression

SUBMITTED IN PARTIAL FULFILLMENT OF THE DEGREE OF

MASTER OF SCIENCE IN BIOTECHNOLOGY

HAKIM WASIM

CENTRE FOR BIOTECHNOLOGY                                                                                

JAMIA HAMDARD (HAMDARD UNIVERSITY)

NEW DELHI-62

UNDER THE SUPERVISION OF

PROF. JITENDRA P.KHURANA

HEAD, DEPARTMENT OF PLANT MOLECULAR BIOLOGY

UNIVERSITY OF DELHI, SOUTH CAMPUS

NEW DELHI

INDIA


ABBREVIATIONS

ºC                                                        Degree centigrade

cDNA                                                         Complimentary DNA

Cm                                                          Centimeter

dATP                                                      Deoxy adenosine 5’-triphosphate

dCTP                                                      Deoxy cytidine 5’-triphosphate

dGTP                                                 Deoxy guanidine 5’triphosphate

dTTP                                                      Deoxy thymidine 5’triphosphate

dsDNA                                                   Double stranded DNA

DMSO                                                    Dimethyl sulfooxide

EDTA                                                    Ethylene diammenotetraacetic acid

EtBr                                                    Ethidium bromide

IPTG                                                      Isopropyl-β-D-thiogalactosidase

Kb                                                          Kilobase

kDa                                                        Kilo Dalton

λ                                                             Lambda

LB                                                          Luria Bertani

LMP                                                       Low melting point

M                                                           Molar

mM                                                        Milli molar

MCS                                                      Multiple cloning site

µl                                                           Microliter

Min                                                        Minutes

mRNA                                                   Messanger RNA  

ng                                                          Nanogram

ORF                                                       Open reading frame

PCR                                                   Polymerase chain reaction

Pfu                                                          Plaque forming unit

RO                                                         Reverse osmosis (Water)

RT                                                      Room temperature

Rpm                                                       Revolution per minute

SDS                                                       Sodium dodecyl sulphate

Sec                                                         Second

SSC                                                       Saline sodium citrate

TAE                                                      Tris acetate EDTA

TE                                                         Tris EDTA

Tris-Cl                                                  Tris buffer (pH adjusted with Concentrated HCl)

UV                                                       Ultra violet

X-gal                                                    5-Bromo-4 chloro-3 indoly-β-D-Galactoside                                                      

                                 

 

ACKNOWLEDGEMENTS

At this moment putting down a few explicit words of thanks in my acknowledgement is a very difficult task for me since the depth of my feelings sometimes is too tough to be arrested in few words.

First and foremost I take this opportunity to thank my supervisor Professor J.P.Khurana whose magnanimous attitude and generous   approach that gave me judicious guidelines with humane touch to accomplish my project.

I will not lag behind in paying regards to my Honorable ‘s Head   of the centre for Biotechnology, Professor P.S Srivastava whose headship combined with scholarship and guardianship not only impacted knowledge but awakened interest for the pursuit excellence.

I also wish to express my grateful to my esteemed faculty Prof.S.K. Jain, Dr M.Z. Abdin, Dr.Fahrat Afrin, Dr. Depsika Pande, Dr. Farha Anjum and Dr. Sandeep Das for their valuable suggestions, support and encouragement.

I am highly indebted to Mr.Mukesh Jain for inculcating me all those qualities, which make him so meticulous, and for training my work and me untiring at each and every stage got an appreciable order due to helping hands provided by Mr.Mukesh jain. And he deserves special thanks for helping a great deal in my concluding Experiment.

Next I express my profound sense gratitude to Dr. Ritu, Mithu, Jithendra Thakur, and Laju Paul for their noble guidance and effective co-operative.  

INTRODUCTION

        

        The two polynucleotide of the parent helix are wound round one another, this means that progression of the replication require the double helix not to just to be unzipped but also to be un wound.  This infects a significant problem if we consider that the E.coli DNA molecule is 4000k.b or 400000 turns of helix in length and must be replicated in twenty minute. The implication that the double helix is rotating at a rate of 6500rpm! This is so inconceivable that for many years molecular biologist s sought solution that avoid unwinding around 1979 things became so desperate that double helix was incorrect and in fact the polynucleotide in double stranded DNA molecule are laid side by side and not wound round each other. Fortunately a group of enzyme that solved the topological problem was eventually discovered.

DNA TOPOISOMERASES

        DNA topoisomerases are a class of enzyme involved in the regulation of DNA super coiling. DNA topoisomerases fall in to two classes Type I and Type II. Both unwind DNA molecules without actually rotating the double helix. Type I topoisomerases change the degree of super coiling of DNA by causing single stranded break and religation, where type II topoisomerases cause double stranded break. Different roles of topoisomerase I and II indicate an opposing pair of roles in the regulation of DNA super coiling. Both activities are especially crucial during DNA transcription and replication when the DNA helix must be unwound to allow proper function of large enzymatic machinery and topoisomerases have indeed been shown to maintain both transcription and replication.

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DNA TOPOISOMERASE I

Structure; The determination of crystal structure of a 67kDa and terminal fragment of E.coli topoisomerase I represents how these enzymes function.

Str-1

The 590 aa N-terminal fragment observed in the structure corresponds to the cleavage/strand passage domain. E.coli topo I contains four domains of protein.

Str-2

 MECHANISM OF DNA RELAXATION

 

                This mechanism has been referred to as enzyme-bridging model for DNA relaxation. The crystal structure of the 67kDa fragment of E.coli enzyme suggests how such reaction occurs. ...

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