lambda – requirements for cloning vectors

2 problems with using wild type lambda as cloning vector

1. few unique restriction endonuclease (RE) sites

– 48.5 kb genome – multiple sites for most enzymes

fixed in several ways:

– recombination between different lambdoid phages

lambda is part of family of closely related phages

– same genome arrangement

– minor differences in DNA sequence

– cause differences in restriction map

different versions of lambda

– infected in same host at same time

– recombinant progeny produced

– some have desired restriction map

mutagenesis of lambda

– mutants with inactivated RE sites screened for

random or site-directed mutagenesis

replacement of non-essential genes

– with different DNA sequences

– e.g. sequences containing multiple cloning sites

2. size requirements for packaging

– distance between cos sites

– must be 78%-105% of wild type lambda genome

if > 2.4 kb cloned into full-length lambda vector

– too long to be packaged

solution – lambda vectors shorter than wild type lambda genome

3 parts of lambda genome not essential for lytic growth (Fig. 3.9)

b-region – region between genes J and N – contains:

att site

– recombination site for lambda & E. coli genomes

int, xis genes

– needed for recombination with E. coli genome

gam gene – needed for rolling circle replication

– lambda packages double-sized circular genomes

–with 2 cos sites

– created by recombination

– between 2 circular lambda genomes

– requires either:

Red (lambda recombination enzyme)

recA⁺ host cell & chi sites in vector

chi – crossover hotspot instigator

– hotspot for RecA-mediated recombination

– do not naturally occur in lambda

– but can be cloned in

cI gene – needed for lysogeny

2.8 kb region between P & Q genes can be deleted

(nin deletion – N-independent)

– lytic growth without N gene product

– allows N gene to be deleted

– total length that can be deleted – up to 24.6 kb

– so 26 kb of DNA could be cloned in

3 types of lambda-based cloning vectors

1. insertion vectors

– contain single site for cloning DNA inserts

– designed to be packaged with/without inserted DNA

– minimum size for packaging – 38 kb

– so can only clone in 10 kb (or less) inserts

uses – cloning small fragments

– cloning cDNA – in expression vectors

examples:

lambda gt11 – (Fig. 3.11) – has complete lacZ gene cloned in it

– blue plaques (with X-gal, IPTG)

– if insert cloned into unique EcoRI site

– get insertion inactivation – colourless plaques

– also is expression vector

– if cDNA cloned in frame with lacZ gene

– get expression of fusion protein in lambda-infected cells

– screen for proper cDNA clone

– using antisera for desired protein

potential problems: – insert must be in frame

– antisera must recognise protein produced in E. coli

– proper folding, glycosylation may be problem

– fusion protein must not be lethal

– kills cells before progeny phage produced

lambda ZAP – (Stratagene) – (Fig. 3.13)

– has Bluescript plasmid cloned in it

– cDNAs cloned in Bluescript multiple cloning site

– get insertion inactivation of lacZ ' gene

(in lacZΔM15 host)

– get fusion protein with lacZ ' gene

– multiple cloning site

– allows cloning in 3 reading frames

– better chance of expression

note – not all sites can be used

– some duplicated in lambda vector

– lacI^q – overexpressed lac operon repressor

– helps prevent expression of toxic fusion proteins

– until operon induced with IPTG

– get automatic subcloning into plasmid vector – Bluescript

– Bluescript in lambda ZAP vector

– cut between f1/M13 initiator & terminator sites

– if E. coli coinfected with:

lambda ZAP vector containing cloned gene

M13 helper phage

– single-stranded DNA replication

– starts and stops at ends of Bluescript

– single-stranded plasmid packaged in M13 coat

– infect host cell

– repaired to double-stranded DNA

– maintained as plasmid

lambda gt10 (Fig. 3.11)

– allows screening using insertion inactivation of cI gene

– cI gene in vector has unique EcoRI site

– cloned insert prevents lysogeny

– only lytic growth – clear plaques

– without insert – lysogens survive in plaques

– turbid plaques

– screen using hfl mutant host

– no Hfl protease, no cleavage of cII gene product

– lambda ⁺ and lambda gt10 without insert always lysogenize

– if cI insertion inactivated – no lysogeny – plaques

2. substitution vectors

– contain 2 cloning sites flanking central “stuffer” fragment

– “stuffer” fragment contains:

enough DNA for minimum packaging length vector

– vector “arms” without stuffer (or cloned insert)

– too short to be packaged

selection or screening system for cloned inserts

use – cloning large DNA fragments – up to 26 kb – genomic DNA libraries

examples:

Charon 4a

– has lacZ gene on EcoRI stuffer fragment

– blue/white screening

lambda EMBL 3 & lambda EMBL 4 – have 3 RE sites flanking stuffer

lambda EMBL 3

left arm^__SalI BamHI EcoRI^_stuffer^_EcoRI BamHI SalI^__right arm

– different order in lambda EMBL 4 (Fig. 3.11)

– EcoRI on outside, SalI on inside

cloning – double digest vector with BamHI & EcoRI

– get arms with BamHI sites, stuffer with EcoRI ends

– precipitate DNA with ethanol

– large fragments recovered

– bits in between sites

– too small to precipitate efficiently – lost

– end up with stuffer that cannot religate to arms

– only cloned DNA inserts make packagable product

selection – vector allows Spi^– selection

– Spi^– phenotype – not sensitive to P2 inhibition

– lambda ⁺ and lambda EMBL with stuffer – have red & gam genes

– will not grow in cells lysogenized by phage P2

– if stuffer replaced with cloned DNA

– red & gam genes lost

– phage will grow in P2 lysogen strain

– must also have recA⁺ host cell & chi sites in vector

– for packaging

Lambda DASH (Stratagene) – has Spi^– selection

– has multiple cloning sites

– T3 and T7 promoters flanking stuffer

– used for “chromosome walking”

– assemble map of overlapping genomic DNA clones

– use library of overlapping DNA fragments

– cloned in Lambda DASH

– use T3 and T7 promoters to make labelled RNA probes

– homologous to ends of 1 clone

– probe other clones

– if probes hybridize, clones overlap end of first clone

– assemble map of cloned fragments

3. cosmids – plasmid containing phage cos site (Fig. 3.14)

– if cosmids arranged so 2 cos sites 38-52 kb apart

– packaged in phage capsids by helper phage

– phage particle infects target cell

– injects cosmid DNA

– replicated as plasmid in cell

– cosmids usually ~ 5 kb long

– 33-47 kb insert allows packaging in capsid

– some cosmids come in “sets” of different sizes

– allow cloning of different-sized fragments

uses – cloning very large fragments of genomic DNA

problems:

– requires concatemer for packaging

– cosmid-large insert-cosmid

– solution – vectors with 2 cos sites

– single large insert cloned in cosmid

– get 2 cos sites far enough apart to be packaged

– self-ligation of vector

– creates product long enough to be packaged

– solution – alkaline phosphatase treatment of vector

– stops self-ligation

– stability of vectors with cloned inserts

– 40-50kb plasmids with pMB1 ori – not very stable

– large size – slows replication – low copy number

– easy to lose vector (unless antibiotic selection used)

– if recombination happens

– between repeated sequences in cloned insert

– smaller derivative grows faster

– takes over culture

solution – use recA mutant hosts