Packaging lambda vectors in vitro

packaging DNA in phage capsids requires:

packagable DNA

phage heads & tails

enzymes

ATP

– if all present, DNA “automatically” packaged

packagable DNA – 2 cos sites 78 %-105 % lambda genome apart

– need concatemer (Fig. 3.12) or circular multimer

– monomer not packaged

– concatemer produced by ligating: left arm phage genome

right arm phage genome

cloned insert

– restriction endonuclease (RE) cut ends & cut cos sites

– have single-stranded “sticky” ends – can be ligated

– ligation conditions

– ideal conditions derived empirically

– usually ~ 2 X as many arms as insert

– high concentrations DNA

– more intermolecular ligation

(concatemers)

– less intramolecular ligation (monomers)

Note:

– insertion vectors can package (unless phosphatased)

right arm(cos)left arm(RE site)right arm(cos)left arm

– sequence between cos sites

packaging extract – contains: phage heads & tails

enzymes

ATP

– prepared from lambda lysogen strains

lysogen strain should have:

mutants in RE genes – no RE in lysate

(cloned DNA not cleaved)

mutants in red gene (phage lysogen) and recA (host)

– no rearrangement of cloned DNA

– no recombination between packaging strain lambda lysogen genome & vector

cI⁸⁵⁷ mutant – temperature sensitive repressor

– lysogen stable at 32ºC – at 40ºC cI inactivated

– lysogen induced to lytic growth

– allows precise control of induction

Sam7 mutant – amber nonsense mutant

– stops natural lysis of cells

– infected cells become “bag of phage particles”

– cells concentrated by centrifugation

– lysed by chloroform

– concentrated packaging extract

problem:

How do you stop lysogen genome from being packaged?

traditional approach:

– use 2 strains mutant in different head protein genes

– 1 strain has amber nonsense mutation in D

– other strain has amber nonsense mutation in E

– each strain alone cannot make functional capsids

– unless host is amber suppressor

– so lysogen genome not packaged

– mix lysates of both strains – get complementation

– wild type D & E proteins assemble into head

– DNA can now be packaged

– lysogen genomes still present in mix

– but can be inactivated with UV radiation

– damages nucleic acids, not proteins

– mix lysates in presence of ligated vectors

– packaging starts

New approach:

use lysogen strain mutated in cos site

– lysogen cannot package its own genome

– only need 1 strain to make extract

Note: preparation of packaging extracts

– time consuming, difficult to optimize

solution: commercial kits – reasonably priced

– optimized by manufacturer

after packaging:

titrate phage: find yield of phage particles produced

amplify library:

infect host cells with packaged recombinant phage

– prepare large scale phage lysate

– now have multiple copies of each clone in library

– less likely to miss desired clone

titrate amplified library

set up screening plates: 50-500 plaques per plate

– screen by plaque lift

– transfer plaques to nylon membrane

– screen with labelled nucleic acid probe