Vectors subjective assignment 2
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This document provides information about vectors used in molecular cloning. It defines vectors as DNA molecules that can carry foreign genetic material into host cells. The main types of vectors discussed are plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are the most commonly used vectors and can exist in various forms like Ti, F, R, and virulence plasmids. Bacteriophages and viral vectors are also described. The document examines the features of vectors, including origins of replication and selectable markers. It concludes with feedback acknowledging the academic writing course.
Vectors subjective assignment 2
- 1. Vectors Prepared By: Pramitkumar Natvarlal Patel Application Number of Course (AW): fb2dbb07e45911e988099313432890b1 Affiliation: Research Scholar at Mehsana Urban Institute of Science, Ganpat University, Kherva, Mehsana, Gujarat
- 2. Outline Definition Introduction Types of Vectors Features of Vectors Questions CC BY-SA-NC 2
- 3. Definition In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g.- plasmid, cosmid, Lambda phages). or A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism. CC BY-SA-NC 3
- 4. Introduction A cloning vector is a DNA molecule in which foreign DNA can be inserted or integrated and which is further capable of replicating within host cell to produce multiple clones of recombinant DNA. Examples: Plasmids, phage or virus A vector containing foreign DNA is termed recombinant DNA. The most commonly used vectors are plasmids. Insertion of a vector into the target cell is usually called transformation for bacterial cells, transfection for eukaryotic cells although insertion of a viral vector is often called transduction. CC BY-SA-NC 4
- 5. Types of Vectors 1. Cloning Vectors Propagation or cloning of DNA insert inside a suitable host cells. Obtaining millions of copies. Examples: Plasmids, Phage or Virus. 2. Expression Vectors Express the DNA insert producing specific protein. Origin of replication. Antibiotic resistance gene. Expression vectors with strong promoters. CC BY-SA-NC 5
- 6. Agents Used as Vectors Plasmids Bacteriophages Cosmid Artificial chromosome vectors In 1973, Cohen described first successful construction of recombinant vector. Plasmid PSC101 – E.coli CC BY-SA-NC 6
- 7. Plasmids Plasmid can be defined as: An extra chromosomal self-replicating structure found in bacterial cells that carries genes for a variety of functions not essential for cell growth. OR A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA is called plasmid. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells. CC BY-SA-NC 7
- 8. Types of Plasmids Conjugative/transmissible vectors: They mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids. Non-conjugative vectors: They do not mediate DNA through conjugation, e.g., many R and col plasmids. CC BY-SA-NC 8
- 9. Types of Plasmids 1. Ti Plasmid A Ti or tumor inducing plasmid is a circular plasmid is often, a part of the genetic equipment of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Use to transduce its genetic material to plants. Important in the creation of transgenic plants. 2. Col Plasmid Any plasmid that carries genetic information for the production of a colicin; Involved in killing the other harmful strains of bacteria also called bacteriocinogenic plasmid. CC BY-SA-NC 9
- 10. Types of Plasmids 3.Virulence Plasmid Virulence plasmid turns the bacterium into a pathogen So they are responsible for carrying the genes which cause diseases. 4. F Plasmid The Fertility factor allows genes to be transferred from one bacterium to another by conjugation. F plasmid a conjugative plasmid found in F+ (male) bacterial cells that leads with high frequency to its transfer. A cell possessing the F plasmid (F+, male) can form a conjugation bridge (F pilus) to a cell lacking the F plasmid (F−, female), through which genetic material may pass from one cell to another.CC BY-SA-NC 10
- 11. Types of Plasmids 5. Degradative Plasmid These plasmid are types of plasmids present in certain bacteria’s such as Pseudomonas putida which impart the ability of degrade xenobiotic compounds. For example: salicylic acid, 2-4,D etc. 6. R (Resistance) Plasmid R plasmid is a conjugative factor in bacterial cells. Promotes resistance to agents such as antibiotics, metal ions, ultraviolet radiation, and bacteriophage. • Many R-factors can pass from one bacterium to another and through which antibiotic resistance spreads between bacterial species, genera and even families. CC BY-SA-NC 11
- 12. Types of Plasmids 7. Yeast Plasmid Yeast naturally harbours plasmid. Yeast cloning vectors include: Yeast Integrative Plasmid and Yeast Replicative Plasmid CC BY-SA-NC 12
- 13. Questions 1. Which of the following is true for a plasmid Property? a) Contains an origin of replication b) Imparts a useful characteristic to the host bacterium c) Possesses one or more genes d) Replicates only when the host genome is undergoing replication 2. How can conjugative and non-conjugative plasmids be differentiated? a) On the basis of size b) Presence of antibiotic resistance c) Number of cloning and digestion sites d) Presence of transfer gene 3. Which of the following is not a type of plasmid? a) F b) R c) Ti d) T4 CC BY-SA-NC 13
- 14. Answer 1. Answer: d Explanation: A plasmid replicates independently of the host genome and this is the property exploited in cloning. All other options given are characteristic features of a plasmid. 2. Answer: d Explanation: Transfer genes present on the conjugative plasmids can help them differentiate from the non-conjugative plasmids. 3. Answer: d Explanation: T4 is a bacteriophage, a virus which is also a desirable cloning vector just like a plasmid. T4 can be used in higher organisms and has a lysogenic life cycle and hence a vector of choice CC BY-SA-NC 14
- 15. Bacteriophages or Viral Vectors A viral vector is a virus which has been modified in a laboratory environment for purpose of introducing genetic material into a cell. To form a viral vector, remove the genes in the virus that cause disease. Then replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact. Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells. But to improve safety, they need to be replication defective so the viruses can be used as vehicles to carry 'good' genes into a human cell. CC BY-SA-NC 15
- 16. Bacteriophages or Viral Vectors The process of delivering genetic material with the use of a virus is known as transduction. What are main types of viral vectors? Retrovirus Adenovirus Lentivirus Adeno-Associated virus (AAV) Herpes virus CC BY-SA-NC 16
- 17. Cosmid Combine parts of the lambda chromosome with parts of plasmids. Contain the cos sites of λ and plasmid origin of replication. Behave both as plasmids and as phages. Cosmids can carry up to 50 kb of inserted DNA. Structure of Cosmid Origin of replication (ori). Restriction sites for cleavage and insertion of foreign DNA. Selectable marker from plasmid. A cos site - a sequence yield cohesive end (12 bases). Ampicillin resistance gene (amp). CC BY-SA-NC 17
- 18. Artificial Chromosome Linear or Circular YACs and BACs can carry a DNA fragment up to 300,000 nucleotides long. Different types Bacterial Artificial Chromosome (BAC) Yeast Artificial Chromosome (YAC) P1 derived artificial chromosome (PAC) Mammalian Artificial Chromosome (MAC) Human Artificial Chromosome. (HAC) YAC – Cloning in yeast BAC & PAC – Bacteria MAC & HAC – Mammalian & Human cells.CC BY-SA-NC 18
- 19. Features of Vectors Origin of replication Promoter Cloning site Genetic markers Antibiotic resistance Epitope Reporter genes Targeting sequence Protein purification tags CC BY-SA-NC 19
- 20. Questions 1. Any DNA molecule that has the ability to replicate in an appropriate host cell, to which the desired gene are integrated for cloning, is called as… a) Plasmid b) linker c) vector d) adapter 2. Which of the following statement is true? a) a vector should have an origin of replication b) a vector should have selectable markers c) a vector should have unique restriction sites d) all of these 3. Expression vectors differ from a cloning vector in having… a) an origin of replication b) suitable marker genes c) unique restriction sites d) control elements 4. Extra chromosomal, double stranded, circular DNA molecule present in bacteria which is widely used as vector is called… a) phagemid b) cosmid c) plasmid d) bacterial vectors CC BY-SA-NC 20
- 21. Questions 5. The first engineered plasmid vector is… a) pBR 322 b) pUC vectors c) pSC101 d) pUC 19 6. Ti plasmids that is used as a plant vector is obtained from… a) Agrobacterium tumefaciens b) Agrobacterium rhizhogenes c) Agrobacterium radiobactor d) Thermus aquaticus CC BY-SA-NC 21
- 22. Answer 1) C (Vector) 2) D (All of these) 3) D (Control elements) 4) C (Plasmid) 5) C (pSC101) 6) A (Agrobacterium tumefaciens) CC BY-SA-NC 22
- 23. Acknowledgement Academic Writing (15 WEEK) course held by UGC (University Grant Commission). Course Co-Ordinator: Dr. Ajay Semalty, H.N.B. Garhwal University (A Central University) Srinagar Garhwal (Uttarakhand). 23
- 24. Feedback of the Course AW Here, I want to put my heartiest gratitude towards all the faculty member who have taught the lessons in this course titled by Academic writing (AW). I, really feel honoured to write this feedback about the course because whatever I have learnt from 15 weeks of lessons, It is an extraordinary boost of my knowledge and very solid step of my research career. It is an outstanding effort by UGC and our respected faculty Dr. Ajay semalty. Without his contribution and endless efforts it is not possible to compete lot many of things we have faced in this course journey. We people are very glade to have you sir in this course!! CC BY-SA-NC 24
- 25. References Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000). "DNA Cloning with Plasmid Vector. Molecular Cell Biology (4th ed.). New York: W. H. Freeman. Acquaah G (16 August 2012). Principles of Plant Genetics and Breeding. John Wiley & Sons Inc. ISBN 978-1-118-31369-5. Johnston C, Martin B, Fichant G, Polard P, Claverys JP (March 2014). "Bacterial transformation: distribution, shared mechanisms and divergent control". Nature Reviews. Microbiology. 12 (3): 181–96. doi:10.1038/nrmicro3199. PMID 24509783. MeSH Browser. meshb.nlm.nih.gov. Retrieved 2018-04-16. Hartl DL, Jones EW (1998). Genetics: principles and analysis (4th ed.). Sudbury, Mass.: Jones and Bartlett Publishers. ISBN 978-0-7637- 0489-6. OCLC 45730915. del Solar, Gloria; Giraldo, Rafael; Ruiz-Echevarría, María Jesús; Espinosa, Manuel; Díaz-Orejas, Ramón (June 1998). "Replication and Control of Circular Bacterial Plasmids. Microbiology and Molecular Biology Reviews. 62 (2): 434–464. ISSN 1092- 2172. PMC 98921. PMID 9618448. Brown TA (2010). "Chapter 2 - Vectors for Gene Cloning: Plasmids and Bacteriophages". Gene Cloning and DNA Analysis: An Introduction (6th ed.). Wiley-Blackwell. ISBN 978-1-4051-8173-0. CC BY-SA-NC 25
- 26. Further reading Freshney IR (2005-07-29). Culture of Animal Cells: A manual of basic technique. Hoboken, New Jersey: John Wiley & Sons, Inc. ISBN 978-0-471-45329-1. CC BY-SA-NC 26