Animal Biotechnology Lec 4 Vectors PDF

Animal biotechnology Lec 4 Vectors Vectors  Cloning Vectors A vector is used to amplify a single molecule of DNA into many copes. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replicat...

Animal biotechnology Lec 4 Vectors Vectors  Cloning Vectors A vector is used to amplify a single molecule of DNA into many copes. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell. Most vectors are genetically engineered plasmids or phages. There are also cosmid vectors, bacterial artificial chromosomes, and yeast artificial chromosomes.  Cloning Vectors A DNA molecule that can carry foreign DNA into a cell and replicate there Plasmids Bacteriophages Cosmids Others Yeast Artificial Chromosomes (YACs) Plant Cloning Vectors Mammalian Cell Vectors  Types of vectors Maximum insert size (kilobases or kb [1000bp]) Bacterial plasmid 6-12 Bacteriophage 25 Cosmids 35 Bacterial artificial chromosome 300 Yeast artificial chromosome 200-1000  Cosmids Engineered hybrids of phage DNA and plasmids that are used for large DNA fragments. YAC Bacterial plasmids Most bacterial DNA is on a single large chromosome, but some DNA is in a small circle called a plasmid. Bacterial Plasmids in Nature Plasmids are circular, double- stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells. usually carry genes that are useful but not essential to survival There can be as many as several hundred copies of a single plasmid in each bacteria. They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb Size and copy number  ‫‪Plasmid Isolation from Bacteria‬‬ ‫تنميه المزرعة البكتيرية المحتوية على البﻼزميد على بيئة سائلة‬ ‫ ‬ ‫جمع الخﻼيا منها بالطرد المركزى‬ ‫ ‬ ‫تحضير المستخلص الخلوى من هذة الخﻼيا )‪(Cell extract‬‬ ‫ ‬ ‫ويتم التخلص من البروتينات‬ ‫ ‬ ‫ باﻻضاﻓة الى ذلك يجب ﻓصل ‪ DNA‬البﻼزميد عن الكميات الكبيرة‬ ‫من ‪ DNA‬الكرموسومات البكتيرية الموجودة ايضا ﻓى الخﻼيا‪.‬‬ Alkaline denaturation  Features of Plasmids used as vectors in gene cloning (Most plasmids are genetically engineered) Small size Origin of replication Multiple cloning site (MCS) Selectable marker genes Have sequences (Promoter) that allow RNA polymerase to transcribe genes  Features of Plasmids used as vectors in gene cloning (Most plasmids are genetically engineered) Small size: are plasmids that have been genetically engineered to reduce unneeded DNA small enough to be isolated without undergoing degradation during purification Can hold up to 10 kb fragments  Features of Plasmids used as vectors in gene cloning Multiple cloning site (MCS) have several unique restriction sites for cloning a DNA fragment so that the vector will cut only once (MCS)  Several restriction sites Each plasmid have different MCS  Features of Plasmids used as vectors in gene cloning Origin of replication: so that the DNA can be replicated within a host cell the region where DNA strands are melted to initiate the replication process  Features of Plasmids used as vectors in gene cloning Promoter: that allow RNA polymerase to transcribe genes  Features of Plasmids used as vectors in gene cloning Selectable marker genes have selectable markers for determining whether cloning vehicle has been transferred into cells and to indicate whether the foreign DNA has been inserted into the vector.  Selectable markers for cellular Screening Growing Culture transformed cells can grow on Blue white screening Gene regulation (Lac Operon) β - galactosidase (z) β - galactoside permease (y) Structural genes galactoside transacetylase (a)  ‫‪ -١‬نظام أوبرون الﻼكتوز في بكتريا الـ ‪E. coli‬‬ ‫‪Lac Operon‬‬ ‫أوﻻً‪ :‬ﻓﻲ ﺣﺎﻟﺔ غيﺎب سكر اﻟﻼكتوز‪:‬‬ ‫ﻻ يﺣدث نسخ ﻟلجينﺎت‬ ‫اﻟتركيبيﺔ‬ ‫‪A- Lactose absent, repressor active, operon off.‬‬  ‫‪ -١‬نظام أوبرون الﻼكتوز في بكتريا الـ ‪E. coli‬‬ ‫‪Lac Operon‬‬ ‫ثﺎنيﺎ ً‪ :‬ﻓﻲ ﺣﺎﻟﺔ وجود سكر اﻟﻼكتوز‪:‬‬ ‫يﺣدث نسخ ﻟلجينﺎت‬ ‫اﻟتركيبيﺔ‬ ‫‪B- Lactose present, repressor inactive, operon on.‬‬