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Title: Co-generating synthetic parts toward a self-replicating system

Journal Article · · ACS Synthetic Biology
 [1];  [2];  [3];  [1];  [4];  [3];  [2];  [1]
  1. Harvard Medical School, Boston, MA (United States); Wyss Harvard Institute of Biologically Inspired Engineering, Boston, MA (United States)
  2. Harvard Medical School, Boston, MA (United States)
  3. Univ. of Florida, Gainesville, FL (United States)
  4. Northwestern Univ., Evanston, IL (United States)
+ Show Author Affiliations

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE, might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when co-expressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in co-expression pool varied from ~1.5 to 5-fold compared to the batch mode, with up to ~ 2.4 µM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ~13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. As a result, this work also points to what is still needed to obtain self-replicating synthetic ribosomes in-situ in the PURE system.

Research Organization:
Harvard Medical School, Boston, MA (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
FG02-02ER63445
OSTI ID:
1348832
Journal Information:
ACS Synthetic Biology, Vol. 6, Issue 7; ISSN 2161-5063
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 31 works
Citation information provided by
Web of Science

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In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme journal May 2010
RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA journal July 2008
Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system posted_content January 2017
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Cited By (14)

Mirror‐Image 5S Ribonucleoprotein Complexes journal January 2020
Gene Expression Inside Liposomes: From Early Studies to Current Protocols journal April 2019
Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors journal August 2018
Templated Self‐Replication in Biomimetic Systems journal April 2019
Towards Design of Self‐Organizing Biomimetic Systems journal March 2019
Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system journal January 2019
The hallmarks of living systems: towards creating artificial cells journal August 2018
Is Research on “Synthetic Cells” Moving to the Next Level? journal December 2018
Mirror‐Image 5S Ribonucleoprotein Complexes journal January 2020
Strategies for in vitro engineering of the translation machinery journal November 2019
Gene-Expressing Liposomes as Synthetic Cells for Molecular Communication Studies journal January 2019
Bacterial cell-free expression technology to in vitro systems engineering and optimization journal June 2017
In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli journal March 2020
MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification journal December 2017

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Related Subjects

60 APPLIED LIFE SCIENCES
cell-free protein synthesis
continuous-exchange
self-replicating system
proteomics
ribosome