In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSU0 that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.
Escherichia coli-based traditional cloning methods use restriction enzyme cleavage followed by DNA joining with DNA ligase (1). These methods are often inefficient and laborious, particularly for the cloning of multiple DNA fragments or a large DNA fragment. On the other hand, yeast cells are an ideal tool for conducting efficient in vivo recombinational cloning (2). Because most animal, insect, and bacterial vectors carry no yeast replication origins, researchers have previously been limited to using yeast-based in vivo cloning procedures for the construction of vectors in experiments involving yeast; for example, yeast two-hybrids (3,4). In order to clone a desired DNA fragment into the vectors lacking yeast replication origins, the yeast-based in vivo cloning method usually requires two rounds of DNA manipulation. In the first round, the vector is converted to a yeast/ E. coli shuttle vector by integrating the replication origin and a selectable marker for yeast using an E. coli-based traditional cloning method. In the second round, the desired DNA fragment is PCR-amplified with 20–40 bp of homology to a yeast vector (2,5) and is cotransformed with the linearized vector into yeast.
To eliminate these two rounds of lengthy DNA manipulation, we developed a new yeast-based in vivo cloning method. Many cloning vectors are derived from the pBR322 plasmid and contain a common backbone sequence carrying an ampicillin resistance (Ampr) gene and a pBR322-derived replication origin for E. coli (pBR322 origin) (6). We constructed a helper plasmid pSU0 that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. Cotransformation of pSU0 into yeast with a pBR322-derived vector and the desired DNA fragment results in the conversion of the pBR322-derived vector into a yeast/ E. coli shuttle vector and simultaneously allows the cloning of the DNA fragment into the same vector. In addition, we show that this method is applicable to multifragment cloning (7). Our new method can replace lengthy traditional cloning methods with an efficient one-step protocol.Materials and Methods Strains
E. coli strain DH5α was used for the propagation of plasmids, and the strain BL21 Star™ (DE3) (Invitrogen, Carlsbad, CA, USA) was used for the expression of recombinant proteins. Yeast strain YPH499 (MATa ura3-52 lys2-801amber ade2-101orchre trp1-Δ63 his3-Δ200 leu2-Δ1) was used for homologous recombination.Construction of pSU0
The helper plasmid pSU0 (GenBank® accession no. AB215109; (Figure 1)A) was constructed by yeast homologous recombination. This plasmid has an Ampr gene, a 2µ yeast replication origin (2µ origin), a URA3 selectable marker, and a pUC-derived replication origin for E. coli (pUC origin) ((Figure 1)A). There is a single nucleotide difference between the pBR322 and pUC origins (8). First, to rearrange these origins and genes in the desired order, they were PCR-amplified from pYES2/CT (Invitrogen). For the PCR amplification of the Ampr gene, 2µ origin- URA3, and the pUC origin, the primer pairs used were pSU0-1 and pSU0-4 (15 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min using Pfu DNA polymerase; Fermentas,Vilnius, Lithuania), pSU0-2 and pSU0-5 (40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 3 min using Pfu DNA polymerase), and pSU0-3 andpSU0-6 (15 cycles of 95°C for 30 s, 55°C for 30s, and 72°C for 1 min using Pfu DNA polymerase), respectively ((Table 1)). The primer pSU0-1 was designed to provide EcoRI and BamHI sites. Next, in order to provide the long overlapping sequence to these fragments, two adjacent fragments were joined by the PCR-based overlap extension method (9) as follows. Using PCR, the Ampr and 2µ origin of replication- URA3 selectable marker fragments, 2µ origin- URA3 and the pUC origin fragments, and the pUC origin and Ampr fragments were joined by the pSU0-1 and pSU0-5, pSU0-2 and pSU0-6, and pSU0-3 and pSU0-4 primer pairs, respectively (45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 4 min using Pfu DNA polymerase). Cotransformation of these three DNA fragments into yeast (10) resulted in homologous recombination at the overlapping sequences and the consequent formation of the plasmid pSU0. The proper formation of pSU0 was confirmed by restriction analysis (data not shown). For the cloning experiments, the plasmid pSU0 was digested with BamHI and EcoRI and purified by electrophoresis.