MISSA is a highly efficient in vivo DNA assembly method for plant multiple-gene transformation

Abstract

We describe a highly efficient in vivo DNA assembly method, multiple-round in vivo site-specific assembly (MISSA), which facilitates plant multiple-gene transformation. MISSA is based on conjugational transfer, which is driven by donor strains, and two in vivo site-specific recombination events, which are mediated by inducible Cre recombinase and phage lambda site-specific recombination proteins in recipient strains, to enable in vivo transfer and in vivo assembly of multiple transgenic DNA. The assembly reactions can be performed circularly and iteratively through alternate use of the two specially designed donor vectors. As proof-of-principle experiments, we constructed a few plant multigene binary vectors. One of these vectors was generated by 15 rounds of MISSA reactions and was confirmed in transgenic Arabidopsis (Arabidopsis thaliana). As MISSA simplifies the tedious and time-consuming in vitro manipulations to a simple mixing of bacterial strains, it will greatly save time, effort, and expense associated with the assembly of multiple transgenic or synthetic DNA. The principle that underlies MISSA is applicable to engineering polygenic traits, biosynthetic pathways, or protein complexes in all organisms, such as Escherichia coli, yeast, plants, and animals. MISSA also has potential applications in synthetic biology, whether for basic theory or for applied biotechnology, aiming at the assembly of genetic pathways for the production of biofuels, pharmaceuticals, and industrial compounds from natural or synthetic DNA.

Figure 1.

Schematic diagram for the MISSA system composed of engineered E. coli strains and vectors. The donor strains were engineered to contain the necessary trans-acting factors (Pir replication initiation protein and Tra conjugational transfer proteins) for replication and conjugational transfer of the suicidal donor vectors. The recipient stains are able to inducibly express two sets of site-specific recombination proteins: Cre recombinase and phage site-specific recombination proteins, including integrase (Int), excisionase (Xis), and integration host factor (IHF). When the donor and recipient strains are mixed together, the donor vector will be transferred into the recipient strain and then site-specific recombination will occur between the donor and recipient vectors. Two rounds of MISSA are shown (A and B). Each round of in vivo site-specific recombination includes two recombination events: the donor vector first integrates into the recipient vector by Cre/loxP-mediated recombination, and then the backbone of the donor vector is removed by the λ phage site-specific recombination event. Since, in the former rounds of recombination, the site-specific recombination sites attR1 (A) and attL2 (B) that can be used for subsequent rounds of recombination are introduced, the recombination reactions can be performed circularly and iteratively. The boxes on the donor and recipient circles represent functional DNA elements, and the colored circles represent protein factors. Functional molecules of the same type, including trans-acting protein factors and cis-acting DNA elements, are indicated with the same colors. The wavy lines represent gene or operon mRNA.

Figure 2.

Schematic diagram of MISSA vectors and recombination reactions based on these vectors. A, Structure of the vectors. The recipient vectors containing the loxP-attR2 cassette are derived from either TAC (P1 ori) or BIBAC (F ori), which exist in E. coli as a single copy and are characterized by high cloning capacity for foreign DNA. Two sorts of donor vectors are used alternately: the pL series of donor vectors contains the loxP-attL1-attL2 cassette, in which the expression cassettes are inserted between the attL1 and attL2 sites; the pR series of donor vectors contains the loxP-attR2-attR1 cassette, in which the expression cassettes are inserted between the attR2 and attR1 sites. B, First round of MISSA. First, the donor vector is integrated into the recipient vector by a Cre/loxP-mediated recombination event. The second in vivo site-specific recombination event specifically occurs between the attL2 and attR2 sites and is mediated by phage site-specific recombination proteins. C, Second round of MISSA. The second round of MISSA is similar to the first, with the only difference being that the second recombination event occurs specifically between attR1 and attL1. Ap, Ampicillin; Cm, chloramphenicol; Glu, Glc; Kn, kanamycin; LB, left border; RB, right border. [See online article for color version of this figure.]

Figure 3.

Schematic diagram of an improved version of the MISSA vectors as well as recombination reaction events based on these vectors. A, The main differences of the improved version of the donor vectors, pLC series and pRG series compared with the original ones (Fig. 2), are that two sorts of antibiotic resistance genes are used and the genes are inserted between the loxP and attL1/attR2 sites to enhance the selection efficiency of recombination clones. B and C, The recombination reactions are similar to those in Figure 2 except that different selection media are used for recombination clones. Two representative rounds of recombination are shown. Ap, Ampicillin; Cm, chloramphenicol; Glu, Glc; Gm, gentamycin; Kn, kanamycin. [See online article for color version of this figure.]

Figure 4.

Physical maps of original cloning donor vectors and recipient vectors. A, Original cloning donor vectors. The functional donor vectors can be acquired by replacing the stuff DNA (pUC_ori-ccdB cassette) on the cloning donor vectors pL/R-ccdB and pLC/RG-ccdB with functional DNA via transitional cut-and-ligate methods. The pPcB/pPrcB vector can be used to obtain functional pL/R series of donor vectors with genes of interest via Gateway BP cloning. The cloning donor vectors are also compatible with TA cloning, and donor T vectors can be prepared by digestion with AhdI. Since the ccdB gene is lethal to most E. coli strains, including the donor strains used in this report, the donor vectors carrying the pUC_ori-ccdB cassette should be propagated in gyrA462 hosts such as DB3.1, in which the pUC origin from the pUC_ori-ccdB cassette is in charge of replication of the donor vectors. After the pUC_ori-ccdB cassette was replaced by genes, gene expression cassettes, or functional DNA elements, the functional donors without the pUC origin were propagated in BW23474 for cloning purposes or in BW20767 for conjugational transfer. T1 and T2, rrnB T1 and T2 transcription terminators; T7/SP6/M13-47/RV-M, universal sequencing primers. B, Recipient vectors. The only differences between the two TAC-derived and the two BIBAC-derived vectors are the positions and directions of the loxP and attR2 sites relative to the T-DNA borders. The key features of the original cloning donor vectors and their derivatives and recipient vectors are indicated under the physical maps.

Figure 5.

Phenotypes of transgenic Arabidopsis and expression levels of transgenes under normal and cold induction conditions. A, Structural features of the T-DNA region of pABA-oriT. The promoters used for each gene can be found in Table I. B, The phenotypes of three representative lines of the T2 generation are shown. The seeds of the wild type (WT; ecotype Columbia) and three transgenic lines were sown on MS agar plates, and the photographs were taken after the seeds germinated and grew for 7 d at 22°C under a 16-h/8-h light/dark cycle. The normal green seedlings in lines 5 and 6 were identified with the segregated wild type based on GUS staining. C to J, Real-time RT-PCR analysis of transcript levels of the T3 generation of transgenic seedlings under normal and cold induction conditions. The relative transcript level was calculated as fold difference from the Actin transcript level, which was used as the internal control; relative expression levels were calculated and normalized with respect to the genes expressed in the untreated wild type.