Total synthesis of Escherichia coli with a recoded genome

Fredens, Julius; Wang, Kaihang; Torre, Daniel de la; Funke, Louise F. H.; Robertson, Wesley E.; Christova, Yonka; Chia, Tiongsun; Schmied, Wolfgang H.; Dunkelmann, Daniel; Beránek, Václav; Uttamapinant, Chayasith; Llamazares, Andres Gonzalez; Elliott, T. S. J.; Chin, Jason W.

doi:10.1038/s41586-019-1192-5

Cited by 394 publications

(353 citation statements)

References 53 publications

(95 reference statements)

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“…The fission spacers are encoded in the plasmid pKW3_MB1Amp_Tracr K _Spacer (Data file S3), which contains a pMB1 origin of replication, an ampicillin resistance marker, the spacer array under the control of its endogenous promoter as previously described (7), and a tracrRNA upstream of the spacer array (5).…”

Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

“…Fission and fusion experiments were performed in the E. coli strain MDS42 (1), which has been utilized for recent genome engineering projects (5). The MDS42 rpsLK43R strain (7) was used throughout our experiments with K43R mutation introduced into the rpsL gene to confer resistance to streptomycin in the absence of an additional wild-type copy of rpsL and sensitivity to streptomycin in the presence of any additional copy of wildtype rpsL.…”

Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

“…Similarly, the recipient strain of the chimeric genome generation experiment contains a watermarked region 2 with the same recoding scheme between coordinates 436,924-939,332 (Data file S6). Both of these strains were generated by GENESIS as described in detail previously (5). The resulting strain for watermarked region 1 contained an rpsL-Kan R cassette at position 1,979,778-1,979,783, and the resulting strain for watermarked region 2 contained a sacB-Cm R cassette at position 1,050,809-1,050,814these were removed from the genome by lambda-red mediated recombination prior to genome rearrangement experiments.…”

Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

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Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

Wang

Torre

Robertson

et al. 2019

Science

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The design and creation of synthetic genomes provide a powerful approach to understanding and engineering biology. However, it is often limited by the paucity of methods for precise genome manipulation. Here, we demonstrate the programmed fission of the Escherichia coli genome into diverse pairs of synthetic chromosomes and the programmed fusion of synthetic chromosomes to generate genomes with user-defined inversions and translocations. We further combine genome fission, chromosome transplant, and chromosome fusion to assemble genomic regions from different strains into a single genome. Thus, we program the scarless assembly of new genomes with nucleotide precision, a key step in the convergent synthesis of genomes from diverse progenitors. This work provides a set of precise, rapid, large-scale (megabase) genome-engineering operations for creating diverse synthetic genomes.

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Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

Section: Strains and Plasmids Used In This Studymentioning

confidence: 99%

See 1 more Smart Citation

Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

Wang

Torre

Robertson

et al. 2019

Science

Self Cite

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“…More recently, in a remarkable technical feat, the total synthesis of an E. coli genome was achieved. This was done specifically to afford a better application of this technology, freeing rare codons for the incorporation of unnatural amino acids (Uaas) [11].…”

Section: Introductionmentioning

confidence: 99%

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Chemla

Ozer

Alfonta

et al. 2019

Preprint

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Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thioboc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

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“…Yet, a rapid method for whole organelle genome engineering for eukaryotic algae has not been established.The first hurdle to overcome is the need for a fast and inexpensive method for synthesizing, assembling, and storing synthetic organelle genomes. While processes for synthesizing and replacing chromosomes have been established for some prokaryotes [30][31][32][33][34] and eukaryotes [35] ,…”

mentioning

confidence: 99%

Rapid method for generating designer algal mitochondrial genomes

Cochrane

Brumwell

Soltysiak

et al. 2019

Preprint

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With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria.HIGHLIGHTSTAR-cloned mitochondrial genome of P. tricornutum in yeastDeveloped PCR-based cloning method to create designer algal mitochondrial genomesStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts

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Total synthesis of Escherichia coli with a recoded genome

Cited by 394 publications

References 53 publications

Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Rapid method for generating designer algal mitochondrial genomes

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