Standardized Representation of Parts and Assembly for Build Planning.

The design and construction of genetic systems, in silico, in vitro, or in vivo, often involve the handling of various pieces of DNA that exist in different forms across an assembly process: as a standalone "part" sequence, as an insert into a carrier vector, as a digested fragment, etc. Communication about these different forms of a part and their relationships is often confusing, however, because of a lack of standardized terms. Here, we present a systematic terminology and an associated set of practices for representing genetic parts at various stages of design, synthesis, and assembly. These practices are intended to represent any of the wide array of approaches based on embedding parts in carrier vectors, such as BioBricks or Type IIS methods (e.g., GoldenGate, MoClo, GoldenBraid, and PhytoBricks), and have been successfully used as a basis for cross-institutional coordination and software tooling in the iGEM Engineering Committee.

CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology.

Multipart and modular DNA part libraries and assembly standards have become common tools in synthetic biology since the publication of the Gibson and Golden Gate assembly methods, yet no multipart modular library exists for use in bacterial systems. Building upon the existing MoClo assembly framework, we have developed a publicly available collection of modular DNA parts and enhanced MoClo protocols to enable rapid one-pot, multipart assembly, combinatorial design, and expression tuning in Escherichia coli. The Cross-disciplinary Integration of Design Automation Research lab (CIDAR) MoClo Library is openly available and contains promoters, ribosomal binding sites, coding sequence, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from that of previously published protocols.

Owl: electronic datasheet generator.

Owl ( www.owlcad.org ) is a biodesign automation tool that generates electronic datasheets for synthetic biological parts using common formatting. Data can be retrieved automatically from existing repositories and modified in the Owl user interface (UI). Owl uses the data to generate an HTML page with standard typesetting that can be saved as a PDF file. Here we present the Owl software tool in its alpha version, its current UI, its description of input data for generating a datasheet, its example datasheets, and the vision of the tool's role in biodesign automation.

Interactive assembly algorithms for molecular cloning.

Molecular biologists routinely clone genetic constructs from DNA segments and formulate plans to assemble them. However, manual assembly planning is complex, error prone and not scalable. We address this problem with an algorithm-driven DNA assembly planning software tool suite called Raven (http://www.ravencad.org/) that produces optimized assembly plans and allows users to apply experimental outcomes to redesign assembly plans interactively. We used Raven to calculate assembly plans for thousands of variants of five types of genetic constructs, as well as hundreds of constructs of variable size and complexity from the literature. Finally, we experimentally validated a subset of these assembly plans by reconstructing four recombinase-based 'genetic counter' constructs and two 'repressilator' constructs. We demonstrate that Raven's solutions are significantly better than unoptimized solutions at small and large scales and that Raven's assembly instructions are experimentally valid.

Thalassiosira spp. community composition shifts in response to chemical and physical forcing in the northeast Pacific Ocean.

Diatoms are genetically diverse unicellular photosynthetic eukaryotes that are key primary producers in the ocean. Many of the over 100 extant diatom species in the cosmopolitan genus Thalassiosira are difficult to distinguish in mixed populations using light microscopy. Here, we examine shifts in Thalassiosira spp. composition along a coastal to open ocean transect that encountered a 3-month-old Haida eddy in the northeast Pacific Ocean. To quantify shifts in Thalassiosira species composition, we developed a targeted automated ribosomal intergenic spacer analysis (ARISA) method to identify Thalassiosira spp. in environmental samples. As many specific fragment lengths are indicative of individual Thalassiosira spp., the ARISA method is a useful screening tool to identify changes in the relative abundance and distribution of specific species. The method also enabled us to assess changes in Thalassiosira community composition in response to chemical and physical forcing. Thalassiosira spp. community composition in the core of a 3-month-old Haida eddy remained largely (>80%) similar over a 2-week period, despite moving 24 km southwestward. Shifts in Thalassiosira species correlated with changes in dissolved iron (Fe) and temperature throughout the sampling period. Simultaneously tracking community composition and relative abundance of Thalassiosira species within the physical and chemical context they occurred allowed us to identify quantitative linkages between environmental conditions and community response.

An end-to-end workflow for engineering of biological networks from high-level specifications.

We present a workflow for the design and production of biological networks from high-level program specifications. The workflow is based on a sequence of intermediate models that incrementally translate high-level specifications into DNA samples that implement them. We identify algorithms for translating between adjacent models and implement them as a set of software tools, organized into a four-stage toolchain: Specification, Compilation, Part Assignment, and Assembly. The specification stage begins with a Boolean logic computation specified in the Proto programming language. The compilation stage uses a library of network motifs and cellular platforms, also specified in Proto, to transform the program into an optimized Abstract Genetic Regulatory Network (AGRN) that implements the programmed behavior. The part assignment stage assigns DNA parts to the AGRN, drawing the parts from a database for the target cellular platform, to create a DNA sequence implementing the AGRN. Finally, the assembly stage computes an optimized assembly plan to create the DNA sequence from available part samples, yielding a protocol for producing a sample of engineered plasmids with robotics assistance. Our workflow is the first to automate the production of biological networks from a high-level program specification. Furthermore, the workflow's modular design allows the same program to be realized on different cellular platforms simply by swapping workflow configurations. We validated our workflow by specifying a small-molecule sensor-reporter program and verifying the resulting plasmids in both HEK 293 mammalian cells and in E. coli bacterial cells.

Mouse intestine selects nonmotile flhDC mutants of Escherichia coli MG1655 with increased colonizing ability and better utilization of carbon sources.

D-gluconate which is primarily catabolized via the Entner-Doudoroff (ED) pathway, has been implicated as being important for colonization of the streptomycin-treated mouse large intestine by Escherichia coli MG1655, a human commensal strain. In the present study, we report that an MG1655 Deltaedd mutant defective in the ED pathway grows poorly not only on gluconate as a sole carbon source but on a number of other sugars previously implicated as being important for colonization, including L-fucose, D-gluconate, D-glucuronate, N-acetyl-D-glucosamine, D-mannose, and D-ribose. Furthermore, we show that the mouse intestine selects mutants of MG1655 Deltaedd and wild-type MG1655 that have improved mouse intestine-colonizing ability and grow 15 to 30% faster on the aforementioned sugars. The mutants of MG1655 Deltaedd and wild-type MG1655 selected by the intestine are shown to be nonmotile and to have deletions in the flhDC operon, which encodes the master regulator of flagellar biosynthesis. Finally, we show that DeltaflhDC mutants of wild-type MG1655 and MG1655 Deltaedd constructed in the laboratory act identically to those selected by the intestine; i.e., they grow better than their respective parents on sugars as sole carbon sources and are better colonizers of the mouse intestine.