Bacteriophage Vectors PDF

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Summary

Notes on lecture delivered by David Groth...

Description

Bacteriophage Vectors Bacteriophages Bacteriophages are bacterial viruses that infect their hosts by attaching to a specific receptor protein on the surface of the bacteria Once attached they are internalised by either:  Direct injection of the DNA  Normal cell processes Infectious particles released through either budding (M13) or lysis (lambda) Types:  M13  Lambda  P1 M13 Based Vectors M13 is a group of filamentous bacteriophages specific for certain strains of E coli Their genome is about 6.4kbp and is enclosed in protein After adsorption onto the bacterium the phage enters the cell where it is converted from the single stranded particle form into the double stranded replicative (Rf) form The Rf form serves as a template for the ssDNA particle form which is extruded from the cell without causing lysis Require bacteria with F’ Large inserts which tend to be unstable Phage Display Display of peptides on the surface of the M13 phage particle Powerful tool for looking at protein-protein interactions Used for “panning” Phagemids Phagemid vectors are plasmids which have been artificially manipulated so as to contain a small segment of the filamentous phage genome They permit successful cloning of inserts several kbp long E. coli are transformed with a recombinant phagemid and super-infected with helper phage which provides the filamentous protein coat λ Bacteriophages Bacteriophages (λ) are used as cloning vectors for the following applications:  Genomic DNA libraries  cDNA libraries The reasons why we use them are:  Very efficient at cloning large fragments (up to 23kbp)  Uses an alternative method of getting the DNA inside the cell  Size limitation (max size of vector plus insert is about 52kbp) Types of λ Vectors Phases of λ lifecycle are the Lytic cycle and Lysogenic cycle Two main types of λ vectors are Replacement and Insertional

Replacement λ Vectors Utilise red and gam genes and Spi selection The red and gam genes promote lysogeny The red and gam genes are carried on a ‘stuffer’ region Strains of λ which contain red and gam genes (Spi + - sensitive to pro-phage inhibition) do not grow well on bacteria which contain a P2 pro-phage (a related phage) often called P2 lysogens Examples are EMBL3/4 The vectors allow 10-20kbp to be inserted Insertional λ Vectors cI and cro in λ are two mutually antagonistic genes In the lytic state the cro dominates and represses cI. Whereas in the lysogenic state the cI dominates and supresses cro and other transcriptional proteins Insertional vectors have a MCS in the cI gene which allow insertion of foreign DNA rsulting in cI disruption Once the cI is disrupted the lytic state is favoured resulting on clear plaques. Nonrecombinants λ have turbid plaques These types of vectors cannot take very large inserts (1-5kbp) Baculoviruses Baculovirus expression system is based upon Autographa californica Nucleopolyhedrovirus (AcMNPV) isolated from the larva of the alfalfa looper to infect insect cells AcMNPV uses many of the protein modification, processing and transport systems present in higher eukaryotic cells The virus that can be propagated to high titers and adapted for growth in suspension cultures to obtain large amounts of recombinant protein with relative ease and cost. Baculovirus are non-infectious to vertebrates and their promoters are inactive in mammalian cells Genome is covalently closed circular double stranded of 134 kbp, due to its small it can accommodate large fragments of foreign DNA Baculovirus Pathogenicity 3 days p.i.:  loss of appetite  lethargy 6-11 days p.i.:  cuticle rupture  ~109 viruses per larvae Baculovirus Life Cycle

Insect Cell Expression Viral vector system:  Gene of interest is expressed using a Baculovirus  Late acting baculovirus promotors control gene transcription  Infection of insect cells or larvae results in foreign gene expression and production of viral particles  Two methods of generating the recombinant baculoviruses pFAST BAC System

Transposition

pFastBac

Plasmid Vector System Foreign gene inserted into a plasmid vector which is then transfected into insect cells Insect or viral promotors control the foreign gene expression Transient or stable expression possible pFast Multibac This vector allows the expression at least 8 different proteins Used to analyse protein complexes and the subunit interactions Multi-step procedure:  Gene A & B are inserted into the vector followed by a double digest with Pme I and Avr II  Gene C & D are inserted into the vector followed by a double digest with BstZ 171 and Spe I at the central multiple cloning site  These two enzymes create compatible “sticky” ends with those generated by Pme I and Avr II  The two vectors are then ligated together creating the equivalent to “A”

γ Secretase Complex...