Abstract
As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire synthetic yeast genome (Sc2.0 project), we need to be able to assemble these 3-kb pieces into larger DNA segments or chromosome-sized fragments. This assembly into larger DNA segments is carried out in vivo, using homologous recombination in yeast. We have successfully used this approach to assemble a 40-kb chromosome piece in the yeast Saccharomyces cerevisiae. A lithium acetate (LiOAc) protocol using equimolar amount of overlapping smaller fragments was employed to transform yeast. In this chapter, we describe the assembly of 3-kb fragments with an overlap of one building block (∼750 base pairs) into a 40-kb DNA piece.
Citations
May 15, 2013·Microbial Cell Factories·Niels G A KuijpersPascale Daran-Lapujade
Dec 12, 2013·Journal of Biological Engineering·Bogumil J KarasPhilip D Weyman
Mar 29, 2014·Science·Narayana AnnaluruSrinivasan Chandrasegaran
Feb 11, 2016·Critical Reviews in Biotechnology·Mario Juhas, James W Ajioka
Jun 11, 2014·Nucleic Acids Research·Radha KrishnakumarJohn I Glass
Mar 11, 2017·Science·Sarah M RichardsonJoel S Bader
Oct 5, 2017·Nucleic Acids Research·Tyson R ShepherdAnthony C Forster
May 2, 2014·Nature Methods·Sriram Kosuri, George M Church
Jun 17, 2015·Genome Biology·Narayana AnnaluruSrinivasan Chandrasegaran
Jul 18, 2018·Molecular Systems Biology·Héloïse MullerRomain Koszul
Jun 23, 2020·Annual Review of Biochemistry·Weimin ZhangJef D Boeke
Jul 17, 2020·PLoS Genetics·Valentine MosbachGuy-Franck Richard
May 5, 2016·Yeast·Damien Coudreuse
Jun 19, 2019·Science China. Life Sciences·Ruixin LiXing-Zhong Liu
Nov 5, 2020·Journal of Microbiology and Biotechnology·Xinran XuWen-Bing Yin
Feb 8, 2020·Current Biology : CB·Nuria Cortes-SilvaInes A Drinnenberg
Jul 8, 2021·Nucleic Acids Research·Lucie PoggiGuy-Franck Richard
Nov 7, 2021·Nature Communications·Weidong WangJianxin Ma