Vectors cont.. Pattern of Infection. Lytic cycle. Pattern of Infection. Question. Dr. Dinithi Peiris Dept. of Zoology

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1 Vectors cont.. Dr. Dinithi Peiris Dept. of Zoology 1 2 Pattern of Infection Lytic cycle 3 Pattern of Infection 4 Question What is the unique feature in this life cycle Phages causes lysis & cell death of the host cell 5 6 1

2 Lysogenic cycle 7 8 Question What is the unique feature in this life cycle Prophage DNA incorporated in host DNA Phage conversion Specialized transduction 9 Types of Bacteriophages 10 Lambda Bacteriophage Lambda genome is approximately 49 kb in length. Many types but 2 are used λ (Lambda) Only 30 kb is required for lytic growth. Thus, one could clone 19 kb of foreign DNA. M13 Packaging efficiency 78%-100% of the lambda genome

3 13 14 Lambda cloning vectors COS site: Cohesive sticky ends Protein capsule result in Lysis tight constraint of Replication amount of DNA fit inside ori it (~ 55kb) It is known that a good portion of lambda waslysogeny not required= Junk DNA Head Tail Circularized lambda Lambda cloning vectors Eliminate the nonessential parts of lambda Filamentous Lysis Replication ori COS Head ss-circular DNA (size 6407 bp) DNA enter in to cell converted to double Tail stranded molecule known as replicative form or Can now insert large pieces of DNA (~ 20 kb) RF. Replicates until there are about 100 copies in the cell

4 How M13 infects and reproduces Uses infects through Pillus Protein coat is stripped and ss DNA is converted to double stranded replicative form DNA replicated by rolling circle method New particles assembled 200 particles per infected cell per generation M13 released without lysis No lysis on bacterial lawn, generally do in liquid culture. Cloning Ss DNA for probes, sequencing Phage display technology, M13 will produce foreign protein on surface as part of its protein coat, can use to generate specific antibodies Advantages of using Bacteriophages Question As a cloning vector used for in vitro recombination Its molecular genetics is well known Do you remember how bacteria protect themselves from bacteriophages (foreign DNA)? DNA is efficiently packed into phage particles They have restriction enzymes to cut them up! Larger insert sites 21 Fragments from 30 to 46 kb can be accommodated by a cosmid vector. Hybrid vector: plasmid vector and bacteriophage lambda vector. cos ends. Cosmid Cloning Vectors ori TetR DNA that can insert in to cosmid vectors are limited by the amount that fit into the capsule. Somewhat usable but difficult to maintain 21.5 kb cos EcoRI Cos site is the only requirement for packaging into phage particle

5 Other vectors Cosmids can infect bacteria & replicate in it. Infected cells grow in to normal cells. Cosmids are extracted from bacteria and mixed with restriction endonucleases. Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonucleases.. 25 BACs (Bacterial artificial chromosomes) Large low copy number plasmids (have ori and selectable marker) Can be electroporated into E. coli Useful for sequencing genomes, because insert size kb 26 Other vectors Vectors THERE IS NO PERFECT VECTOR YAC (Yeast Artificial Chromosome) Can be grown in E.coli and Yeast Miniature chromosome (contains ori, selectable markers, two telomeres, and a centromere Can accept 200 kb kb; useful for sequencing Ti plasmids; to introduce genes into plants As much as we try to combine the properties of several vector systems, e.g. λ, there is now no vector that can combine all properties in one molecule. Thus a fine tuning process is necessary to optimize parameters. THE HISTORY OF VECTOR DESIGN IS A BALANCE BETWEEN COMPETING OBJECTIVES!! How is Foreign DNA Inserted in to Cells To open up the DNA a restriction enzyme is used. How is Foreign DNA Inserted in to Cells Cut the DNA at a specific place called a restriction site The result is a set of double-stranded DNA pieces with single-stranded ends 29 These ends that jut out are not only "sticky" but they have gaps that can be now be filled with a piece of foreign DNA For DNA from an outside source to bond with an original fragment, one more enzyme is needed DNA ligase seals any breaks in the DNA molecule 30 5

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