A self-regenerating synthetic cell model

Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

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“…Supporting code for Fig. 3f is available on GitHub at https://github.com/lbnc-epfl, https://doi.org/10.5281/zenodo.4160155 59…”

Section: Data Availabilitymentioning

confidence: 99%

A partially self-regenerating synthetic cell

2020

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“…Functional modification of one or more of the molecules comprising PURE via functions present in the source-cells may contribute to PURE activity yet not be present within PURE. As examples, Li et al reported that E. coli ribosomes consisting of PURE-synthesized 30S r-proteins, native 16S rRNA, and a native 50S ribosomal subunit only had ~13% activity compared to the native E. coli ribosomes [27]; Hibi et al showed that PURE itself could simultaneously remake 21 tRNA, one for each corresponding amino acid and initiator tRNA, and when used by PURE recovered ~40% of full PURE activity [28]; Lavickova et al showed that PURE could regenerate the T7 RNA polymerase (RNAP) and eight tRNA synthetases within a diluting fluidic system initiated by functional PURE [29]; and Libicher et al verified that PURE, through serial transfer, could make functional T7 RNAP, two energy recycling factors, and an ensemble of 12 tRNA synthetases and RF1 [30]. Separately, Libicher et al and Doerr et al used multi-plasmid systems to encode ~30 of the translation factors of PURE and verified successful co-expression via mass spectrometry, but it is unclear whether these PURE-made enzymes were functional [25,31].…”

Section: Figure 1 Can We Build Cells From Lifeless Ensembles Of Independently-sourced Natural Biomolecules? (A)mentioning

confidence: 99%

Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building

Wei¹,

Endy²

2021

Preprint

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The construction of synthetic cells from lifeless ensembles of molecules is expected to require integration of hundreds of genetically-encoded functions whose collective capacities enable self-reproduction in simple environments. To date the regenerative capacities of various life-essential functions tend to be evaluated on an ad hoc basis, with only a handful of functions tested at once and only successful results typically reported. Here, we develop a framework for systematically evaluating the capacity of a system to remake itself. Using the cell-free Protein synthesis Using Recombinant Elements (PURE) as a model system we apply our framework to evaluate the capacity of PURE, whose composition is completely known, to remake 36 life-essential functions. We find that only 23 of the components can be well tested and that only 19 of the 23 can be remade by the system itself; translation release factors remade by PURE are not fully functional. From both a qualitative and quantitative perspective PURE alone cannot remake PURE. We represent our findings via a standard visual form we call the Pureiodic Table that serves as a tool for tracking which life-essential functions can work together in remaking one another and what functions remain to be remade. We curate and represent all available data to create an expanded Pureiodic Table in support of collective coordination among ongoing but independent synthetic cell building efforts. The history of science and technology teaches us that how we organize ourselves will impact how we organize our cells, and vice versa.

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“…By periodically flowing fresh extract we are able to use far smaller reagent volumes (∼3 µL/h), or approximately 20x less than previous methods (∼60 µL/h) [17]. Additionally, on-chip mixing of lysate and energy components eliminates the need to cool the premixed reagents off-chip [25]. Using the multiplexing (MUX) valves ( Fig.…”

Section: Design and Characterization Of The Cfpumentioning

confidence: 99%

CFPU: a cell-free processing unit for high-throughput, automated in vitro circuit characterization

Swank

Maerkl

2020

Preprint

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Forward engineering synthetic circuits is at the core of synthetic biology. Automated solutions will be required to facilitate circuit design and implementation. Circuit design is increasingly being automated with design software, but innovations in experimental automation are lagging behind. Microfluidic technologies made it possible to perform in vitro transcription-translation (tx-tl) reactions with increasing throughput and sophistication, enabling screening and characterization of individual circuit elements and complete circuit designs. Here we developed an automated microfluidic cell-free processing unit (CFPU) that extends high-throughput screening capabilities to a continuous reaction environment, which is essential for the implementation and analysis of more complex and dynamic circuits. The CFPU contains 280 chemostats that can be individually programmed with DNA circuits. Each chemostat is periodically supplied with tx-tl reagents, giving rise to sustained, long-term steady state conditions. Using microfluidic pulse width modulation (PWM) the device is able to generate tx-tl reagent compositions in real-time. The device has higher throughput, lower reagent consumption, and overall higher functionality than current chemostat devices. We applied this technology to map transcription factor based repression under equilibrium conditions and implemented dynamic gene circuits switchable by small molecules. We expect the CFPU to help bridge the gap between circuit design and experimental automation for in vitro development of synthetic gene circuits.

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A self-regenerating synthetic cell model

Cited by 6 publications

References 62 publications

A partially self-regenerating synthetic cell

A partially self-regenerating synthetic cell

Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building

CFPU: a cell-free processing unit for high-throughput, automated in vitro circuit characterization

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