T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis

Abstract

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3'-5' exonuclease activity or together with a 5'-3' exonuclease for its DNA polymerase activity. Here, we present a 'T5 exonuclease DNA assembly' (TEDA) method that only uses a 5'-3' exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

Figure 1.

The schematic of the TEDA method. The blue half-moon represents T5 exonuclease. The double lined rectangle with a gap represents a linearized plasmid. The double vertical lines represent the insert DNA. Lines with same color indicate the homologous region. Step 1: T5 exonuclease cuts from the 5′ ends of linearized plasmid and insert DNA to generate 5′-overhangs. Step 2: the 5′-overhangs anneal to each other. Step 3: The cyclized DNA with DNA gaps is transformed into cells and the gaps are repaired in vivo.

Figure 2.

Enzymes and buffer components required for TEDA. (A) The pKat-eGFP fragment was cloned into SmaI-digested pBluescript SK–. The assembly of the two fragments was used as a model for the test. (B) Taq DNA ligase, Phusion DNA polymerase, T5 exonuclease (T5 exo), NAD⁺ were tested for their necessity for the DNA assembly. In addition, Prime-STAR or FastPfu was also used instead of Phusion for testing; (C) PEG 8000 and dNTPs were further tested for their necessity for the DNA assembly. The concentrations of relevant components mentioned above were indicated in the figure. The base solution contained 0.1 M Tris–HCl (pH 7.5), 10 mM MgCl₂ and 10 mM dithiothreitol. The reaction was processed at 50°C for 1 h, which was the same as the Gibson assembly. *, Gibson; **, Hot Fusion; **, TEDA with dNTPs and at 50°C; ****, TEDA without dNTPs at 50°C. The data are averages of three parallel experiments with STDEV.

Figure 3.

TEDA was used for multiple DNA fragment assembly and SDM at multiple sites. (A) The phbCAB gene cluster was used as ABC, or separated into three fragments (A, B and C), or two fragments (AB and C, or A and BC). These fragments were assembled with SmaI-digested pBluescript SK- (V) by using TEDA. (B) The phbA, phbB and phbC genes on pBBR1MCS2–5Ptac-phbCAB contained one, two, or three stop codons (TAA). The efficiency of TEDA to remove one (1 site SDM for site 1), two (2 sites SDM for sites 1 and 3) or three (3 sites SDM for all 3 sites) stop codons in one single TEDA reaction was tested. The modified QuikChange SDM method was applied to remove one ‘TAA’ codon at site 1 as a control. The data are averages of three parallel experiments with STDEV.

Figure 4.

The effect of host strains on TEDA efficiency. The Inoue method was used to prepare the competent cells. Middle-lacZ and pBBR1MCS5::lacZ-truncated with 15 bp homologous arms were assembled by using TEDA (Supplementary Figure S4B). The TEDA reaction mixture was transformed into competent cells of different E. coli strains (gray columns). As a control, the intact pBluescript SK- plasmid was transformed into the competent cells to test for the transformation efficiency (dark columns). The data are averages of three parallel experiments with STDEV.

Figure 5.

The effect of the linearized vector with different ends on TEDA. (A) The schematic presentation of the linearized vector with different ends and insert containing 20-pb homologous arms. Pkat-eGFP-3′Oh-4bp-plus and Pkat-eGFP-5′Oh-4bp-plus, the 4 bp of the overhangs were homologous to the 20-bp arm of the insert; Pkat-eGFP-3′Oh-4 bp-minus and Pkat-eGFP-5′Oh-4bp-minus, the 4 bp of the overhangs was not homologous to the insert arm. (B) The cloning efficiency with different ends and inserts containing 20-bp arms by using TEDA. The data are averages of three parallel experiments with STDEV.

Figure 6.

Comparison of different assembly methods. (A) TEDA was compared with In-fusion and SLIC for the assembly of two fragments. Middle-lacZ and pBBR1MCS5::lacZ-truncated with 15-bp or 20-bp overlaps were used. 1:1, the same molar ratio of the insert to vector was used for DNA assembly; 1:2, double molar amount of the insert to vector was used for DNA assembly. (B) TEDA was compared with Gibson and non-optimized TEDA methods. The Pkat-eGFP and SmaI-pSK was used for cloning. TEDA(0.04U)−30°C, 0.04 U T5 exonuclease at 30°C for 40 min; TEDA(0.08 U)−30°C, 0.08 U T5 exonuclease at 30°C for 40 min; TEDA(0.04 U)−50°C, 0.04 U T5 exonuclease at 50°C for 40 min; Gibson, 0.08 U T5 exonuclease with Phusion and Taq DNA ligase at 50°C for 60 min. Neg, DNA fragments were transformed without TEDA treatment. (C) TEDA was compared with In-fusion for 4 fragments assembly. The 5Ptac-phbCAB operon was separated into three fragments (Figure 2A), and they were assembled with linearized pBBR1MCS-2 to generate pBBR1MCS2::5Ptac-phbCAB. The data are averages of three parallel experiments with STDEV.