Abstract
Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).
Highlights
Synthetic biology is an emerging field on the borderlines of several scientific disciplines, combining biology with engineering and computer science (Haseloff and Ajioka, 2009)
Based on the results summarized in the graph (Figure 5) we hypothesized that in order to achieve the highest co-expression of two proteins, the best strategy would be to use two independent non-replicating plasmids in a mixture of two Agrobacterium strains
We attempted to combine the advantages of the GB and MoClo systems
Summary
Synthetic biology is an emerging field on the borderlines of several scientific disciplines, combining biology with engineering and computer science (Haseloff and Ajioka, 2009). One of the major challenges in synthetic plant biology is the combinatorial construction of complex multigene structures for functional testing in plant cells. Extended Set of GoldenBraid Vectors such multigene constructs: individual pre-assembled parts (modules) are assembled together using predetermined rules, and these units are in turn used in the assembly of incrementally more complex higher order structures. This principle offers several advantages, such as speed, versatility - i.e., the ability to share already prepared and tested parts between laboratories for new applications – as well as significant combinatorial capabilities and low cost (Ellis et al, 2011)
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