TY - JOUR
T1 - HomeRun vector assembly system
T2 - A flexible and standardized cloning system for assembly of multi-modular DNA constructs
AU - Li, Ming V.
AU - Shukla, Dip
AU - Rhodes, Brian H.
AU - Lall, Anjali
AU - Shu, Jingmin
AU - Moriarity, Branden S.
AU - Largaespada, David A.
PY - 2014/6/24
Y1 - 2014/6/24
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0100948
DO - 10.1371/journal.pone.0100948
M3 - Article
C2 - 24959875
AN - SCOPUS:84903517766
SN - 1932-6203
VL - 9
JO - PloS one
JF - PloS one
IS - 6
M1 - e100948
ER -