Chapter_5_-_Vectors_v2

Chapter_5_-_Vectors_v2 - Vectors 5.1 Plasmid Vector (key...

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Vectors 5.1 Plasmid Vector ( key concept: bacterial vector elements for cloning genes ) Plasmid replication, Cloning sites, Selectable markers, Insertional inactivation, Transformation 5.2 Vectors Based on the Lambda Bacteriophage ( key concept: lambda phage are used for making genomic and cDNA libraries ) Lambda biology - Lysogeny, - Lytic cycle, In vitro packaging, Insertion vectors, Replacement vectors 5.5 Expression vectors ( key concept: key factors for expression in different cell types or producing tagged proteins ) Transcriptional and Translational fusion vectors, Utility of promoters 5.6 Vectors for Cloning and Expression in Eukaryotic Cells ( key concept: eukaryotic expression vectors are also bacteria shuttle vectors ) Yeast, Mammalian cells
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5.1 Plasmid Vectors Properties of plasmid vectors Plasmids are extrachromosomal, circular, double-stranded and supercoiled DNA Gene cloning vectors are typically 3-5 kb Most bacterial vectors are closely related to naturally occurring E. coli plasmid called Co1E1 Able to disseminate antibiotic resistance genes among bacteria
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5.1 Plasmid Vectors 5.1.1 Plasmid replication All enzymes needed for replication are present in the host cell Necessary information in plasmid for replication in host cell is the origin of replication (ORI) most plasmid are present in hundreds of copies per cell Preferable sometimes to use low copy number plasmids if the gene product is harmful to the bacterium Most plasmids are quite restricted in bacterial host range that they will replicate in Often want to clone and purify plasmid DNA from one bacterial strain and then express the gene into a more appropriate host for expression, which may be bacteria or yeast strain or mammalian cell Shuttle vectors contain multiple ORIs, one for directing replication in a standard E. coli intermediate host (DH5alpha, XL1-Blue) and another for replication and expression of the protein in a different, perhaps eukaryotic host
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5.1 Plasmid Vectors 5.1.2 Cloning sites  Unique restriction sites for insertion of foreign DNA into plasmids are cloning sites . Insertion of a BamHI-cut fragment into a cloning site results in two BamHI sites on either side of the cloned fragment in the vector Cloned fragment can insert in either orientation (forward or backward) Figure 5.1 Cloning with a plasmid vector Bla = beta-lactamase (ampicillin resistance), ori= origin of replication
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6.1 Plasmid Vectors 5.1.3 Multiple cloning sites, Selectable markers  Most cloning plasmids contain a multiple cloning site (MCS) with a series of unique restriction sites, enabling directional cloning of fragments cut with two enzymes MCS provides lots of choice in restriction enzymes that could be used for cloning Only 1% of bacterial cells will take up plasmids in highly efficient transformations β-lactamase gene ( bla ), codes for the enzyme hydrolyses β-lactam (penicillin-like)
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_5_-_Vectors_v2 - Vectors 5.1 Plasmid Vector (key...

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