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WHY COSMIDS ARE USED AS VECTORS
(BTC012- CONCEPTS IN BIOTECHNOLOGY) MR. HARSH (lecturer in concepts in biotechnology)
SUBMITTED BY:NAME ROLLNO REG NO : : : DILBAGH SINGH 109X06 1040070143 Btech BIOTECH (154)
PROGRAM : SECTION
# INDEX:1. 2. 3. 4. 5. 6. 7. 8. ACKNOWLEDGEMENT INTRODUCTION WHAT IS BIOTECHNOLOGY? APPLICATION OF BIOTECHNOLOGY WHAT IS GENETIC ENGINEERING? ENGINEERING STEPS WHAT ARE VECTORS? WHAT ARE COSMIDS AND WHY THEY ARE USED AS VECTORS? 9. FEATURES OF COSMIDS 10. BIBLIOGRAPHY
I would like to express my gratitude to all those who gave me the possibility to complete this Term Paper. I want to thank the Department of Biotechnology to complete my term paper in the first instance, to do the necessary research work and to use departmental data. I am deeply indebted to Mr. Harsh, Lecturer in Concepts in Biotechnology whose help, stimulating suggestions and encouragement helped me in all the time of research for and writing of this thesis.
With the rise of field of biotechnology, it is becoming essential to make people aware of what is basically a biotechnology and its respective components. The aim of this report to make people aware about biotechnology, its applications, genetic engineering, genetically modified organisms (GMO’s), cloning vectors (cosmids). A description and an analysis of above components is recommended.
# WHAT IS BIOTECHNOLOGY?
Biotechnology is technology based on biology, basically when used in agriculture, food science, and medicine. Biotechnology may be defined as:
Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.
Biotechnology is used to refer to genetic engineering technology of the 21st century, however the term is used on a wider range and history of procedures for modifying biological organisms according to the needs of humanity, going back to the initial modifications of native plants into improved food crops through artificial selection and hybridization. Bioengineering is the science upon which all biotechnological applications are based. With the development of new approaches and modern techniques, traditional biotechnology industries are also acquiring new horizons enabling them to improve the quality of their products and increase the productivity of their systems.
# APPLICATIONS OF BIOTECHNOLOGY:Biotechnology has applications in four major industrial areas:(1) (2) (3) (4) Health care (medical), Crop production and agriculture, Non food (industrial) uses of crops Other products (e.g. biodegradable plastics, vegetable oil, biofuels).
# WHAT IS GENETIC ENGINEERING?
Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes. Genetic engineering is different from traditional breeding, where the organism's genes are manipulated indirectly; genetic engineering uses the techniques of molecular cloning and transformation to alter the structure. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research.
# ENGINEERING STEPS:1. 2. 3. 4. 5. Isolation of the genes of interest Insertion of the genes into a transfer vector Transfer of the vector to the organism to be modified Transformation of the cells of the organism Separation of the genetically modified organism (GMO) from those that have not been successfully modified
# WHAT ARE VECTORS ?
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (such as phage λ) are most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
pBR322 is a plasmid and one of the most commonly used E. coli cloning vectors. pBR322 was the first artificial plasmid created by Mexicans Bolivar and Rodriguez in 1977 and was named after the scientist duo. p stands for plasmid, BR for Bolivar and Rodriguez.
# WHAT ARE COSMIDS AND WHY THEY ARE USED AS VECTORS?
Cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the Lambda phage. Cosmids can be used to build genomic libraries. Cosmids are able to contain 37 to 52 kbp of DNA, while normal plasmids are able to carry only 1-20 kbp. They can replicate as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transfected cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.
Unlike plasmids, they can also be packaged in phage capsids, which allows the foreign genes to be transferred into or between cells by transduction. Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination. To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends, also known as cos sites. In this way, they are similar to using the lambda phage as a vector, but only that all the lambda genes have been deleted with the exception of the cos sequence.
Cos sequences are ~200 base pairs long and essential for packaging. They contain a cosN site where DNA is nicked at each strand, 12bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is nicking and separating the strands. The cosQ site of next cosmid (as rolling circle replication often results in linear concatemers) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases. Because of the fixed size of the phage head, terminase can only package cosmids that are between 75% and 105% of the length of the normal phage. Thus the practical upper limit of the insert size is around 40kb, since there will also need to be origins of replication, selection genes and multiple cloning sites. To package even more DNA into a vector, bacterial artificial chromosomes or yeast artificial chromosomes can be used.
A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperaturesensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI.
It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilization functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.
# FEATURES OF COSMIDS:The typical features of the cosmids are:1. They can be used to clone DNA inserts of upto 40kb. 2. They can be packaged into λ particles, which infect host cells; this is many fold more efficient than plasmid transformation. 3. Selection for recombinant DNA is based on the procedure applicable to the plasmid making up the cosmid. 4. Finally, these vectors are amplified and maintained in the same manner as the contributing plasmid.
# BIBLIOGRAPHY:1. http://www.google.co.in/ 2. http://en.wikipedia.org/ 3. Biotechnology expanding horizons- B.D.Singh (kalyani publications)