Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.
A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.
Ren
Z J
ZJ
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201-1503, USA.
Baumann
R G
RG
Black
L W
LW
D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D015321:Gene Rearrangement; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D014161:Transduction, Genetic; D014170:Transformation, Genetic; D014764:Viral Proteins
D036022:Capsid Proteins; D004270:DNA, Circular; D004279:DNA, Viral; C108525:HOC protein, T4 phage; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D008025:Ligases