Jonassonhoff4375

Although COMPASS was initially developed for pathway engineering, it can equally be employed for balancing gene expression in other synthetic biology projects.Modular cloning standards based on Golden Gate DNA assembly allow for construction of complex DNA constructs over several rounds of assembly. Despite being reliable and automation-friendly, each standard uses a specific set of vectors, requiring researchers to generate new tool kits for novel hosts and cloning applications. JUMP vectors (Valenzuela-Ortega and French, bioRxiv 799585, 2019) combine the robustness of modular cloning standards with the Standard European Vector Architecture and a flexible design that allows researchers to easily modify the vector backbone via secondary cloning sites. This flexibility allows for JUMP vectors to be used in a wide variety of applications and hosts.Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, robust, and highly accurate DNA assembly method, which provides 99% correct assemblies for a typical four-part assembly, enabling high efficiency cloning workflows (Storch et al., ACS Synth Biol, https//doi.org/10.1021/sb500356 , 2015). BASIC employs standardised DNA linkers to combine bioparts, stored in the universal BASIC format. Once a new biopart is formatted into BASIC standard, defined by flanking 18 bp prefix and suffix sequences, it can be placed at any position and in any context within a designed BASIC assembly. This modularity of the BASIC approach is further enhanced by a range of functional linkers, including genetic elements like ribosomal binding sites (RBS) and peptide linkers. The method has a single tier format, whereby any BASIC assembly can create a new composite BASIC part in the same format used for the original parts; it can thus enter a subsequent BASIC assembly without the need for reformatting or changes to the workflow. This unique idempotent cloning mechanism allows for the assembly of constructs in multiple, conceptionally simple hierarchical rounds. Combined with its high accuracy and robustness, this makes BASIC a versatile assembly method for combinatorial and complex assemblies both at bench and biofoundry scale. The single universal storage format of BASIC parts enables compressed universal biopart libraries that promote sharing of parts and reproducible assembly strategies across labs, supporting efforts to improve reproducibility. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol, relies on a single tier format, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round (Casini et al., Nat Rev Mol Cell Biol. https//doi.org/10.1038/nrm4014 , 2015).Start-Stop Assembly is a multi-part, modular, Golden Gate-based DNA assembly system with two key features which distinguish it from previous DNA assembly methods. Firstly, coding sequences are assembled with upstream and downstream sequences via overhangs corresponding to start and stop codons, avoiding unwanted 'scars' in assembled constructs at coding sequence boundaries. Scars at these crucial, sensitive locations can affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. Start-Stop Assembly is therefore both functionally scarless (an advantage usually only achieved using bespoke, overlap-based assembly methods) and suitable for efficient, unbiased and combinatorial assembly (a general advantage of Golden Gate-based methods). Secondly, Start-Stop Assembly has a new, streamlined assembly hierarchy, meaning that typically only one new vector is required in order to assemble constructs for any new destination context, such as a new organism or genomic location. This should facilitate more rapid and convenient development of engineered metabolic pathways for diverse nonmodel organisms in order to exploit their applied potential. This chapter explains both design considerations and practical procedures to implement multi-part, hierarchical assembly of multi-protein expression constructs, either individually or as combinatorial libraries, using Start-Stop Assembly.Mobius Assembly is a versatile and user-friendly DNA Assembly method, which facilitates rapid and simple generation of DNA constructs. Mobius Assembly combines high cloning capacity and vector toolkit simplicity to streamline combinatorial assemblies. It is a two-level hierarchical modular cloning system that enables quadruple assembly augmentation. It adopts the 4 bp standard overhangs defined by Phytobricks to promote standard part sharing, and it can be made compatible with different chassis. Furthermore, Mobius Assembly reduces domestication requirements and uses chromogenic proteins to facilitate the identification of positive assemblies.Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms. In this method, we describe how to design and construct new Phytobricks as well as how to assemble them in both manual and nanoscale automated one-step reactions. Finally, we describe a high-throughput method for sequence verification of assembled plasmids.Creating DNA constructs is a basic and fundamental step in molecular and synthetic biology. While prices for gene synthesis are decreasing, it is still more economical in most cases to assemble constructs from a library of components (Parts). Many methods for DNA assembly are available, but most require either a fixed and inflexible format for the construct, with all Parts first being cloned in specific donor plasmids, or remaking Parts with new homology ends for each specific assembly reaction, requiring large numbers of single-use oligonucleotides. PaperClip assembly allows Parts stored in any format (linear PCR products or synthetic DNA, or cloned in any plasmid) to be used in totally flexible assembly reactions; up to 11 parts can be assembled in a single reaction, in any order, to give a linear or circular construct, and the oligonucleotides required in the assembly process can be reused in any subsequent assembly. In addition to constructing plasmids for bacterial transformation, PaperClip is also well suited to generate linear products for direct transfection of yeast, mammalian, or cyanobacterial cell lines. Thus, PaperClip offers a simple, flexible, and economical route to multipart assembly of constructs for a wide variety of purposes.DNA assembly methods are essential for multiple applications including synthetic biology. We recently developed MetClo, a method that uses a single type IIS restriction enzyme for hierarchical modular DNA assembly. This offers great flexibility in the design of the assembly experiment and simplicity of execution. Here we describe a protocol for hierarchical assembly of large DNA constructs from modular DNA parts using the MetClo vector set, a set of assembly vectors designed for the MetClo method.Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the Modular Cloning system MoClo. Making constructs using the MoClo system requires users to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. A variety of modular cloning systems have been developed, based on Golden Gate cloning for DNA assembly, to enable users to assemble multigene constructs from libraries of standard parts using a series of successive one-pot assembly reactions. Standard parts contain the DNA sequence coding for a genetic element of interest such as a promoter , coding sequence or terminator . Standard parts for the modular cloning system MoClo must be flanked by two BsaI restriction sites and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of basic parts. This protocol requires the following steps (1) defining the type of basic part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large basic parts, it is preferable to first clone subparts as intermediate level -1 constructs. These subparts are sequenced individually and are then further assembled to make the final level 0 module.High-throughput, inexpensive DNA sequencing is an essential component of large-scale DNA assembly operations. Using traditional and acoustic liquid-handling robotics, Illumina's Nextera Tagmentation reactions can be miniaturized and paired with custom PCR index primers to produce highly multiplexed NGS libraries for pooled sequencing. TRAM-34 nmr This chapter describes a high-throughput protocol that enables the simultaneous sequencing of thousands of DNA constructs in a single sequencing run at a dramatically reduced cost compared to bench-top methods.Yeast homologous recombination is a reliable, low-cost, and efficient method for DNA assembly. Using homology regions as short as 24 base pairs, constructs of up to 12 unique parts can be assembled into a diverse range of vectors. The simplicity and robustness of this protocol make it amenable to laboratory automation and high-throughput operations. Here we describe a high-throughput protocol to generate DNA parts through PCR, assemble them into a vector via yeast transformation, and "shuttle" the resulting plasmid constructs into E. coli for storage and propagation. Though this protocol is intended for high-throughput workflows, it can be easily adapted for bench-scale DNA assembly.This protocol describes a high-throughput approach to PCR for the generation of over a thousand amplicons in parallel. Modern liquid handling robotics are used to accelerate reaction setup, miniaturize reaction volumes, and dramatically reduce reagent and consumable cost. Although the focus is on generating DNA parts for use in DNA assembly techniques, this methodology can be applied to any workflow where the parallel production of hundreds or thousands of PCR amplicons is required.