HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs

Abstract

Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector Assembly System (HVAS), employs a three-tiered vector series that utilizes both multisite gateway cloning and homing endonucleases, with the former building individual functional modules and the latter linking modules into the final construct. As a proof-of-principle, we first built a two-module construct that supported doxycycline-induced expression of green fluorescent protein (GFP). Further, with a three-module construct we showed quantitatively that there was minimal promoter leakage between neighbouring modules. Finally, we developed a method, in vitro Cre recombinase-mediated cassette exchange (RMCE) cloning, to regenerate a gateway destination vector from a previous multisite gateway cloning reaction, allowing access to existing DNA element libraries in conventional gateway entry clones, and simple creation of constructs ready for in vivo RMCE. We believe these methods constitute a useful addition to the standard molecular cloning techniques that could potentially support industrial scale synthesis of DNA constructs.

Figure 1. Schematic illustration of the HVAS vector system and its assembly process.

1A, Assembly of a functional module in pBASE vectors from elements in pENTR vectors; 1B, Assembly of multi-modular construct in pHR assembly vectors from modules in pBASE shuttle vectors. Up to 4 elements in pENTR vectors can be assembled on each pBASE shuttle vector, and up to 4 modules in the pBASE shuttle vectors can sequentially dock onto the pHR assembly vector. Abbreviations: L1, attL1; R5, attR5; L5, attL5, L4, attL4; R4, attR4; R3, attR3; L3, attL3; L2, attL2; R2, attR2; R1, attR1; HE, homing endonuclease; DEST, destination cassette; HE1, I-SceI; HE2, I-CeuI; HE3, PI-SceI; HE4, PI-PspI; ITR, inverted terminal repeat.

Figure 2. Building of a functional two-module construct with HVAS which supported doxycycline-induced GFP expression.

2A, schematic illustration of the construct. The abbreviations of depicted elements were annotated in Table 1. 2B, above construct was stably transfected into HCT116 cells, and selected with 1 µg/ml puromycin. Surviving cells were treated with or without 1 µg/ml doxycycline over night before image acquisition with bright field and fluorescent microscopy.

Figure 3. Minimal cross-modular promoter leakiness was observed with dual luciferase assay.

3A, Schematic illustration of the three-module construct. The abbreviations of depicted elements were annotated in Table 1. 3B, and 3C, response of firefly (3B) and renilla (3C) luciferase activities to increasing concentrations of doxycycline; the above construct was stably transfected into HCT116 cells and selected with 1 µg/ml puromycin. Surviving cells were seeded in 24-well plates the day prior to treatment to reach around 80% confluence at the time of treatment; vehicle or doxycycline of indicated concentrations were added for incubation for 24 hours at 37°C before dual luciferase assay. Each treatment was triplicated. Results were represented as relative light units normalized to protein content (RLU/µg). Comparisons were made between results in samples treated with indicated doxycycline concentration and those treated with vehicle only (doxycycline 0 µg/ml). *, p<0.05; **, p<0.01.

Figure 4. In vitro Cre RMCE cloning and its application in HVAS.

4A, schematic illustration of regeneration of a destination vector with in vitro Cre RMCE cloning. The abbreviations of depicted elements were annotated in Table 1. 4B, fluorescence microscopy for HCT116 cells stably transfected with a 2-module construct built with HVAS and in vitro Cre RMCE cloning as illustrated. Open triangle, canonical loxP; solid triangle, loxN variant. 4C, in vivo RMCE, the cells shown in 4B were transfected with floxed puroR cDNA along with pRN-Cre, selected with both puromycin (1 µg/ml) and G418 (1 mg/ml). Fluorescence and bright field imaging of representative colonies were shown.