Figure 1.
Basic gap-repair cloning procedure.
DNA fragment(s) from a linearized plasmid vector are prepared by restriction enzyme digestion or by PCR. DNA fragment(s) from the insert are amplified by PCR, ensuring that the sequence at the end of the fragment is homologous with that of the plasmid. Both DNA fragments are then simultaneously introduced into the cell. Transformants are selected by identifying the plasmid vector marker. The fragments are connected by the homologous recombination activity that occurs within the cell. The constructed plasmid is recovered from the transformed cell into E. coli to amplify and check the structure of the plasmid with restriction enzyme digestion and DNA sequencing. Cells with plasmids that have the desired structure are used for functional analysis.
Figure 2.
Cloning the LEU2 marker gene using GRC in S. pombe.
(A) Blueprint of the plasmid construction. DNA fragments containing LEU2 genes were amplified by PCR using the indicated primer sets with pRS315 as a template. Homologous regions between the vector plasmid (pDblet) and the insert fragments are shown in the same colors. (B) Result of transformation with the linearized (HindIII-KpnI digested) vector, circular vector, and linearized vector with inserts as shown in A. Transformants of Sp286h+ were selected on –uracil plates (EMM with leucine and adenine). (C) A replica of the transformants on the plate in B on a –leucine plate (EMM with adenine).
Table 1.
Structural verification of plasmids found in the transformants after pDblet + LEU2 construction (Figure 2C).
Figure 3.
Construction of a plasmid containing nmt1 promoter: EGFP using GRC in S. pombe.
(A) Blueprint of the plasmid construction. DNA fragments containing nmt1 promoter and EGFP were amplified by PCR using the indicated primer sets with pHM004_nmt1-1 and pKT128 as templates, respectively. Homologous regions between the vector plasmid (pDblet) and the insert fragments are shown in the same colors. (B) Result of transformation. Transformants of Sp286h+ were selected on –uracil plates (EMM with leucine and adenine). The fluorescent GFP image was superimposed on a bright-field image. (C) Independent colonies from plate B were streaked on –uracil plates. The resulting image is shown in B.
Table 2.
Analysis of independent transformants after pDblet + Pnmt1-EGFP construction (Figure 3C).
Figure 4.
Construction of a plasmid containing nmt1 promoter: EGFP using GRC in the lig4Δ mutant S. pombe.
GRC was performed as in Figure 3 except FY4102 was used as a host strain. Transformants were randomly selected and streaked on –uracil +thiamine plate, then the transformants were replica-plated on +thiamine (A) and –thiamine (B) plates. Negative controls (transformants with the vector) were also streaked. Arrowheads indicate transformant with weak GFP fluorescence, in which GFP was integrated into genome.
Figure 5.
Construction of plasmids with different auxotrophic markers using GRC in S. pombe.
(A) Blueprint of plasmid construction. DNA fragments containing each marker gene and the plasmid backbone were amplified by PCR using the indicated primer sets (the primer names are listed in the Supplementary Information) with 972 h genomic DNA and pDblet as templates, respectively. Homologous regions between the fragments are shown in the same colors. For each marker gene, FY7054 were transformed with 3 PCR fragments (i.e. a marker fragment and 2 plasmid fragments) and the transformants were selected on EMM plates without the amino acid associated with the auxotrophic marker. Plasmids were recovered from the transformants and their structure was checked. (B) A map of the original pDblet. (C–F) Maps of plasmids constructed in this study.