Kk - Bacteriophage Lambda Vectors

Bacteriophage Lambda Vectors

 Most

suitable cloning vehicles for Eukaryotic DNA. (Adv. Over plasmids is high).  Large number of phage plaques – characterization and screening.  In vitro packaging system – DNA packaged into empty phage heads.  Infectivity of rDNA – high, compared to pure DNA preparations.  Gene library – few milli lit. of broth.  E.coli phages such as Φ80, 21, 82, 434, P2.  Lytic and Lysogenic pathway.  100 progeny particles per infected cell.

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Molecular weight – 31 x 106 Da and 48,502 bp dsDNA – linear molecule. 5’ ends - Short complementary sequences -12 nucleotides long – cos sites. Circularisation of DNA afetr infection. 40 genes – functional clusters. Gens (A-J) – Head and Tail proteins. Left side. Central region – int, xis, exo etc.,- responsible for lysogenisation. Not essential for lytic growth – deleted to construct suitable vectors. Genes - Right of central region – Six regulatory genes (cI, cII, cIII, cro, N and Q). O and P – essential for DNA replication – lytic growth. S and R – lysis of cellular membranes.

Genes are clustered by function in the lambda genome Late control Recombination att

int

Pint

Control region Replication

gam red xis cIII N

tL1

cI

cro

cII O P Q

Virus head Lysis &tail SR

PL oL PRM PR tR1 PRE tR2 PR‘ t6S tR3 oR origin

promoter operator terminator

A…J

cos

Not to scale!

General structure - λ vectors          

Regions between J and N – 38 to 68%. 30 x 485 = 14,500 bp of foreign DNA. Also 105% of complementary λ – packaged into phage heads. Some other - Non-essential regions. 24.6 kb of foreign DNA into λ DNA. 105% of DNA (52 kb) – upper limit of packagable DNA. 75% (38 kb) – lower limit. Less than 38 kb – strong selection system. Two types of vectors – Insertional vectors and Replacement vectors. Positive Selection for recombinant phage genomes.

Construction of vector       

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Wild type λ DNA – many restriction sites. Located within essential regions. Distribution of sites – altered. Point mutations, substitutions, deletions – two step method. First – λ mutants which do not contain restriction site for particular enzyme. Second – Desired cleavage sites introduced by genetic crosses. Comparitively easy – eliminate restriction cleavege sites completely – growing phages on restricting hosts. Eg., Phage DNA devoid of Eco RI sites will be able to survive in host cells containing Eco RI rstriction and modification system.

 Restriction-resistant

phages - selected – crossed subsequently with suitable susceptible phages – desired combination of cleavage sites.  Eco RI sites limited system.  λb221c1857 - deletion b221 – removes two Eco RI sites.  First step of selection – derivative with no Eco RI restriction sites.  Recombination with λplac5imm (5 Eco RI sites) – occurs in limited region near third Eco RI site from right.

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λ derivatives – different restriction sites. Bam HI site – insertion of DNA obtained with a number of isochizomeric enzymes. Strategy change – One Bam HI site occurs in essential region. Cannot be easily eliminated by passages on restricting host. Klein and Murray – mutagenesis of phage lysate with nitrous acid. Mutagenised phage population was amplified, progeny phages harvested, DNA isolated and cleaved with Bam HI. Small fraction of Bam HI resistant DNA was isolated, packaged in vitro and propagated in suitable hosts. Desired phage derivatives containing only one or two Bam HI sites – obtained by genetic cross.