Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in ye...Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,p GF,harboring a PHIS3–HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79%indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.展开更多
基金supported by the National Science and Technology Major Project of China(2018ZX10711001-006)the Key Research Program of the Chinese Academy of Sciences(KJZD-SW-L06-02)。
文摘Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,p GF,harboring a PHIS3–HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79%indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.
