Figure 1.
Schematic diagram of IRDL cloning.
A, Construct expression of one insert by IRDL cloning. The purified PCR products of GOI and expression vector are mixed in a single tube together with restriction enzymes 1 and 2, as well as ligase. When the digestion and ligation of the mixture are in balance, two possible ligation products appear: (1) and (2). Only E. coli harbored the desired product (2) by its ability to survive on selection plates after transformation. Since the two different restriction enzymes used will generate two incompatible DNA ends, only the appropriate inserts can be ligated with the expression vector. B, Construction of fusion protein by IRDL cloning. The purified products of GOI1, GOI2, and expression vector are mixed in a single tube together with restriction enzymes 1, 2, and 3, as well as ligase. C, Construction of fusion protein by overlap extension PCR and IRDL cloning. GOI, gene fragment of interest. Lethal, lethal gene.
Figure 2.
Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method.
A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans1-T1 as described in materials and methods. (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.
Table 1.
Efficiency of cloning in pWXY1.0 under different conditions.
Figure 3.
Fusion of two inserts, ScDGA1 and EGFP, into a yeast expression vector pWXY3.0 using IRDL cloning.
A, The plasmid pWXY3.0-ScDGA1-EGFP generated by using IRDL cloning method. B, Colony PCR results from 24 recombinant colonies were run on 1% agarose gels. All of the colonies tested contained the corrected inserts. M, DNA ruler DL2501 (Generay). C, TLC separation of neutral lipids from different yeast strains. The transgenic H1246α with the ScDGA1 and ScDGA1-EGFP restore triacylglycerol (TAG) synthesis compared with negative control (H1246α with pWXY3.0). D, Fluorescence microscopy of ScDGA1-EGFP fusion protein expression in transgenic yeast cells. E, Detection of ScDGA1-EGFP fusion protein by western blotting.
Figure 4.
Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector.
A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with EcoRI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli. B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).