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Genetic Toolkits to Design and Build Mammalian Synthetic Systems

2021; Elsevier BV; Volume: 39; Issue: 10 Linguagem: Inglês

10.1016/j.tibtech.2020.12.007

0167-9430

Roberto Di Blasi, Annalise Zouein, Tom Ellis, Francesca Ceroni,

Gene Regulatory Network Analysis

Synthetic biology benefits from standardisation. The development of the field relies on simple, cheap, and high-throughput methods that improve the design–build–test cycle. The development of high-throughput cloning methods has paved the way to the emergence of many cloning toolkits, providing a wealth of standardised parts that can be easily assembled, thus speeding up the engineering of complex genetic devices. Although cloning toolkits for mammalian synthetic biology are underrepresented compared to their bacterial and yeast counterparts, some are now made available to the community. Construction of DNA-encoded programs is central to synthetic biology and the chosen method often determines the time required to design and build constructs for testing. Here, we describe and summarise key features of the available toolkits for DNA construction for mammalian cells. We compare the different cloning strategies based on their complexity and the time needed to generate constructs of different sizes, and we reflect on why Golden Gate toolkits now dominate due to their modular design. We look forward to future advances, including accessory packs for cloning toolkits that can facilitate editing, orthogonality, advanced regulation, and integration into synthetic chromosome construction. Construction of DNA-encoded programs is central to synthetic biology and the chosen method often determines the time required to design and build constructs for testing. Here, we describe and summarise key features of the available toolkits for DNA construction for mammalian cells. We compare the different cloning strategies based on their complexity and the time needed to generate constructs of different sizes, and we reflect on why Golden Gate toolkits now dominate due to their modular design. We look forward to future advances, including accessory packs for cloning toolkits that can facilitate editing, orthogonality, advanced regulation, and integration into synthetic chromosome construction. nonprofit plasmid repository that distributes plasmids constructs. reaction in which many DNA parts are added together; restriction enzymes facilitate the fitting together of parts into a final construct in a (usually) preset order. standardised DNA parts flanked by specific restriction enzymes (Prefix: EcoRI, XbaI; Suffix: SpeI, PstI) allowing iterative assembly of larger DNA constructs. iterative cycle widely implemented in metabolic engineering. Starting from an initial pool of designs (design), these are built and implemented in the system of interest (build), and their functionality tested (test). The test phase returns valuable information to further better the quality of the designs (learn) which can then be imputed again into the cycle. the process in which a sequence of interest is introduced into a toolkit usually by the addition of prefix and suffix sequences. procedure aimed at modifying the genome with mutations, insertions, or deletions at specific locations. collection of DNA parts (e.g., promoters, genes, and terminators) that can be used as starting material for the cloning of larger constructs long DNA sequences (linear or circular) for assembly of large constructs or the study of chromosomes. modular cloning. A method of making golden gate technology modular. a method with a simplified design stage, as constructs are created from standardised units. This simplifies processes and reduces chances of errors. unwanted and nonspecific changes in the genome sequence that may arise when using gene editing technologies, such as CRISPR-Cas9. having little or no cross-reactivity with other components in a system, referring to sequences in this context. sequence of DNA that is transcribed into RNA molecule. It includes promoters and termination sequences. proteins that bind promoters and alter gene expression output from a transcriptional unit. a MoClo-based toolkit for the high-throughput cloning of constructs for yeast engineering.