bacteriophage as cloning vectors

2 problems with using wild type lambda as cloning vector: 1. size requirements for packaging - distance between cos sites must be 78%-105% of wild type lambda genomeDNA molecule can only be increased in size by about 5% addition of only ~2.4 kb of new DNA. Limits the size of inserts.

What is the solution?

Remove parts of lambda genome not essential for lytic growth basically genes involved in lysogeny total length that can be deleted up to 24.6 kb lambda vectors shorter than wild type lambda genome so 26 kb of DNA could be cloned in These deleted will be non-lysogenic and can only follow the lytic cycle.

2. Few unique restriction endonuclease (RE) sites

genome is large (48.5kb) - has more than one recognition sequence for virtually every RE. Solution? mutagenesis of lambda mutants with inactivated RE sites screened for -random or site-directed mutagenesis

 that lacks certain restriction sites can be isolated by natural selection.

3 types of lambda-based cloning vectors: a) Insertion vectors b) Replacement / substitution vectors c) Cosmids

1. Insertion vectors
- A large segment of the non-essential region has been deleted, and the two arms ligated together. - Possess at least one unique restriction site for insertion of new 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

a) gt10
Examples:

allows screening using insertion inactivation of cI gene cI gene in vector has unique EcoRI site -can carry up to 8 kb of new DNA, inserted into the EcoRI site at the cI gene. -insertional inactivation of this gene will produce recombinants which can be identified as clear rather than turbid plaques. cloned insert prevents lysogeny only lytic growth clear plaques

b) Charon 16 (Blattner et al, 1977)

-carries a complete lacZ gene blue plaques (with X-gal, IPTG) if insert cloned into unique EcoRI site get insertion inactivation colourless plaques

2. Replacement Vectors
-Has two recognition sites for the RE used for
cloning. -The sites flank a DNA segment that is replaced by DNA to be cloned. -a "stuffer" sequence of about 15 kb that contains selectable markers, as well as restriction endonuclease cut sites at both ends -Digestion with the relevant enzyme releases this "stuffer fragment" leaving space for a relatively large insert (up to 26Kb) use cloning large DNA fragments up to 26 kb genomic DNA libraries

 WES. B

-has 2 EcoRI sites that flank the replacement fragment -recombinant selection based on size -inserts up to 15 kb.

b) EMBL4

-has 3 different RE which can be used to remove stuffer fragment. -DNA with variety of sticky ends can be cloned -selection based on size or Spi phenotype -inserts up to 23 kb (near maximum).

Spi selection
lambda + (wild type) and lambda EMBL with stuffer have red & gam genes will not grow in cells lysogenized by phage P2 (Spi+ phenotype-sensitive towards P2 prophage inhibition) if stuffer replaced with cloned DNA red & gam genes lost phage will grow in P2 lysogen strain (has Spi phenotype not sensitive to P2 inhibition) must also have recA+ host cell & chi sites in vector for packaging

EMBL4 can carry up to 23 KB of foreign DNA and it also offers positive selection for recombinants. This is dependent upon the fact that wild-type lambda phage cannot infect a cell lysogenic for the related phage, P2. Such phage are Spi+ (sensitive to P2 inhibition) and in EMBL4, the genes responsible for this phenotype are located on the stuffer fragment. Thus non-recombinant EMBL4 are Spi+ but recombinants are Spi- and only the latter can grow on a host lysogenic for phage P2.

 Cosmids were first developed by Collins and Hohn. Comprised of : Plasmid sequences : origin of replication, one/two selectable markers, unique restriction sites Cos site of phage * Only cos sites are needed by the enzymes that package DNA molecule into the protein coat. Cosmids allow cloning of ~ 40 kb segments of foreign DNA. High molecular weight fragments of foreign DNA are generated by partial digestions are incubated with the linear cosmid molecules (at high DNA concentration) Amongst the various types of ligation products will be molecules in which the 40 kb segment of foreign DNA is flanked by 2 cosmid molecules with the cos sites in the same orientation.

3) Cosmids

 Amongst the various types of ligation products will be molecules in which the 40 kb segment of foreign DNA is flanked by 2 cosmid molecules with the cos sites in the same orientation. In vitro packaging of the recombinant molecule a monomer formed with cohesive termini circularised by the cos sites. After infection of the E. coli cells, the recombinant DNA will replicate as a plasmid and express the drug resistance marker (Ampicillin).

b) Cloning using pJB8

pJB8

 In use, cosmids are linearised by cutting at a unique restriction site and ligating to large fragments of foreign DNA. This forms concatamers of cosmid and insert DNA which, when subjected to in vitro packaging are cleaved at the cos sites and packaged into infective particles. Cells containing cosmids are selected as antibiotic resistant colonies. One advantage of cosmids is that, in principle, all such colonies are recombinant because non-recombinants are too small to be packaged.