000096832 001__ 96832
000096832 005__ 20201124104757.0
000096832 0247_ $$2doi$$a10.1074/jbc.REV119.009109
000096832 0248_ $$2sideral$$a114546
000096832 037__ $$aART-2019-114546
000096832 041__ $$aeng
000096832 100__ $$aWatson, Jake F.
000096832 245__ $$aIn vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning
000096832 260__ $$c2019
000096832 5060_ $$aAccess copy available to the general public$$fUnrestricted
000096832 5203_ $$aMolecular cloning is a cornerstone of biomedical, biotechnological, and synthetic biology research. As such, improved cloning methodologies can significantly advance the speed and cost of research projects. Whereas current popular cloning approaches use in vitro assembly of DNA fragments, in vivo cloning offers potential for greater simplification. It is generally assumed that bacterial in vivo cloning requires Escherichia coli strains with enhanced recombination ability; however, this is incorrect. A widely present, bacterial RecA-independent recombination pathway is re-emerging as a powerful tool for molecular cloning and DNA assembly. This poorly understood pathway offers optimal cloning properties (i.e. seamless, directional, and sequence-independent) without requiring in vitro DNA assembly or specialized bacteria, therefore vastly simplifying cloning procedures. Although the use of this pathway to perform DNA assembly was first reported over 25 years ago, it failed to gain popularity, possibly due to both technical and circumstantial reasons. Technical limitations have now been overcome, and recent reports have demonstrated its versatility for DNA manipulation. Here, we summarize the historical trajectory of this approach and collate recent reports to provide a roadmap for its optimal use. Given the simplified protocols and minimal requirements, cloning using in vivo DNA assembly in E. coli has the potential to become widely employed across the molecular biology community.
000096832 536__ $$9info:eu-repo/grantAgreement/ES/DGA/FEDER$$9info:eu-repo/grantAgreement/ES/MCIU/RTI2018-095629-J-I00
000096832 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000096832 590__ $$a4.238$$b2019
000096832 591__ $$aBIOCHEMISTRY & MOLECULAR BIOLOGY$$b87 / 297 = 0.293$$c2019$$dQ2$$eT1
000096832 592__ $$a2.283$$b2019
000096832 593__ $$aBiochemistry$$c2019$$dQ1
000096832 593__ $$aMolecular Biology$$c2019$$dQ1
000096832 593__ $$aCell Biology$$c2019$$dQ1
000096832 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000096832 700__ $$0(orcid)0000-0002-4254-3148$$aGarcía-Nafría, Javier$$uUniversidad de Zaragoza
000096832 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000096832 773__ $$g294, 42 (2019), 15271-15281$$pJ. biol. chem.$$tJournal of Biological Chemistry$$x0021-9258
000096832 8564_ $$s378028$$uhttps://zaguan.unizar.es/record/96832/files/texto_completo.pdf$$yVersión publicada
000096832 8564_ $$s44306$$uhttps://zaguan.unizar.es/record/96832/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000096832 909CO $$ooai:zaguan.unizar.es:96832$$particulos$$pdriver
000096832 951__ $$a2020-11-22-12:40:26
000096832 980__ $$aARTICLE