File Name: programming cells by multiplex genome engineering and accelerated evolution .zip
Protocol DOI: Generating mutant strains is an essential component of microbial genetics. Natural genetic transformation, a process for the uptake and integration of foreign DNA, is shared by diverse microbial species and can be exploited for making mutant strains.
- Multiplex Genome Engineering Methods for Yeast Cell Factory Development
- Recent advances in genetic engineering tools based on synthetic biology
- Multiplex Automated Genome Engineering (MAGE)
- Conjugative Assembly Genome Engineering (CAGE)
Multiplex Genome Engineering Methods for Yeast Cell Factory Development
However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales 3.
Although in vitro and directed evolution methods 4 , 5 , 6 , 7 , 8 , 9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes.
Here, we describe multiplex automated genome engineering MAGE for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes mismatches, insertions, deletions.
We applied MAGE to optimize the 1-deoxy- d -xylulosephosphate DXP biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC Plant Cell 6 , — Download references. We are grateful to J. Jacobson for his insights and advice throughout this work. We thank D. Court for his insights and sharing strain DY, N. Reppas for advice and sharing strain EcNR2, F. Sterling for assistance in constructing the EcFI5 strain. We also thank M.
Recent advances in genetic engineering tools based on synthetic biology
Metrics details. We present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies. Genome editing and DNA synthesis technologies are enabling the construction of engineered organisms with synthetic metabolic pathways [ 1 ], reduced and refactored genomes [ 2 , 3 , 4 , 5 ], and expanded genetic codes [ 6 , 7 ].
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Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many by multiplex genome engineering. and accelerated evolution.
Multiplex Automated Genome Engineering (MAGE)
Genome-scale engineering is a crucial methodology to rationally regulate microbiological system operations, leading to expected biological behaviors or enhanced bioproduct yields. Over the past decade, innovative genome modification technologies have been developed for effectively regulating and manipulating genes at the genome level. Here, we discuss the current genome-scale engineering technologies used for microbial engineering.
Conjugative Assembly Genome Engineering (CAGE)
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As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed.
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Любой шифр можно взломать - так гласит принцип Бергофского. Она чувствовала себя атеистом, лицом к лицу столкнувшимся с Господом Богом. - Если этот шифр станет общедоступным, - прошептала она, - криптография превратится в мертвую науку.
Но… офицер ничего не сказал о… - Разумеется. Я не сказал ему про спутницу.
Ответа не последовало. Бринкерхофф подошел к кабинету. Голоса показались ему знакомыми. Он толкнул дверь. Комната оказалась пуста.
Ты же всегда стремился к большей ответственности. Вот. Он печально на нее посмотрел. - Мидж… у меня нет никакой жизни. Она постучала пальцем по кипе документов: - Вот твоя жизнь, Чед Бринкерхофф.
Но если держать дистанцию, можно заметить его вовремя.