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New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching's versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist's toolbox.

Synthetic gene circuits emerge from iterative design-build-test cycles. Most commonly, the time-limiting step is the circuit construction process. Here, we present a hierarchical cloning scheme based on the widespread Gibson assembly method and make the set of constructed plasmids freely available. Our two-step modular cloning scheme allows for simple, fast, efficient, and accurate assembly of gene circuits and combinatorial circuit libraries in Escherichia coli . The first step involves Gibson assembly of transcriptional units from constituent parts into individual intermediate plasmids. In the second step, these plasmids are digested with specific sets of restriction enzymes. The resulting flanking regions have overlaps that drive a second Gibson assembly into a single plasmid to yield the final circuit. This approach substantially reduces time and sequencing costs associated with gene circuit construction and allows for modular and combinatorial assembly of circuits. We demonstrate the usefulness of our framework by assembling a CRISPR-based double-inverter circuit and a combinatorial library of 3-node networks.

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly's ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.