scholarly journals A self-regenerating synthetic cell model

2020 ◽  
Author(s):  
Barbora Lavickova ◽  
Nadanai Laohakunakorn ◽  
Sebastian J. Maerkl
Keyword(s):  
Resource Competition ◽  
Cell Model ◽  
Translation System ◽  
System Function ◽  
Fundamental Function ◽  
Dna Templates ◽  
Synthetic Cell ◽  
Synthesis Activity ◽  
Robust System ◽  
Protein Components

AbstractSelf-regeneration is a fundamental function of all living systems. Here we demonstrate molecular self-regeneration in a synthetic cell model. By implementing a minimal transcription-translation system within microfluidic reactors, the system was able to regenerate essential protein components from DNA templates and sustained synthesis activity for over a day. By mapping genotype-phenotype landscapes combined with computational modeling we found that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieved 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.

scholarly journals A partially self-regenerating synthetic cell

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Barbora Lavickova ◽  
Nadanai Laohakunakorn ◽  
Sebastian J. Maerkl
Keyword(s):  
Resource Competition ◽  
Living Systems ◽  
Translation System ◽  
System Function ◽  
Fundamental Function ◽  
Dna Templates ◽  
Synthetic Cell ◽  
Synthesis Activity ◽  
Robust System ◽  
Protein Components

AbstractSelf-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.

scholarly journals Shaping liposomes by cell-free expressed bacterial microtubules

2021 ◽  
Author(s):  
Christophe Danelon ◽  
Marileen Dogterom ◽  
Johannes Kattan ◽  
Anne Doerr
Keyword(s):  
Lipid Membranes ◽  
Cell Biology ◽  
De Novo ◽  
Lipid Vesicles ◽  
Translation System ◽  
Dna Templates ◽  
Cytoskeletal Network ◽  
Synthetic Cell ◽  
Directional Transport ◽  
Shape Changes

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.

An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2)

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3299-3309 ◽  
Author(s):  
Yoshiko Okamoto-Hosoya ◽  
Takeshi Hosaka ◽  
Kozo Ochi
Keyword(s):  
Protein Synthesis ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Translation System ◽  
In The Wild ◽  
Synthesis Activity ◽  
Aberrant Protein ◽  
Ribosomal Protein S12 ◽  
High Level ◽  
Actinorhodin Production

Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.

Trypanosoma brucei mitochondrial ribonucleoprotein complexes which contain 12S and 9S ribosomal RNAs

Parasitology ◽  
1998 ◽  
Vol 116 (2) ◽  
pp. 157-164 ◽  
Author(s):  
H. H. SHU ◽  
H. U. GÖRINGER
Keyword(s):  
Cell Membrane ◽  
Trypanosoma Brucei ◽  
Translation System ◽  
12S Rrna ◽  
Translation Inhibition ◽  
Translational Activity ◽  
Ribonucleoprotein Complexes ◽  
Rnp Complex ◽  
Synthesis Activity ◽  
Translational Apparatus

Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria. The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed. Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane. Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent. We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle. We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts. The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size. This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17·5S, containing the 9S rRNA, and 20S containing the 12S rRNA. A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2[ratio ]3 ratio (20S[ratio ]17·5S).

Isoproterenol induces a ribosomal modification in the heart and the skeletal muscle of hamsters

1985 ◽  
Vol 5 (1) ◽  
pp. 13-19
Author(s):  
Josette Noël ◽  
Léa Brakier-Gingras
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Magnesium Concentration ◽  
Translation System ◽  
Synthesis Activity ◽  
And Control

The protein synthesis activity of heart, skeletal muscle and liver polysomes from isoprotenerol-treated and control hamsters has been compared in an in vitro non-inititating translation system. Heart and skeletal muscle polysomes from treated hamsters were less active than controls and required a higher magnesium concentration for optimal protein synthesis. These results suggest that there is a conformational modification in heart and skeletal muscle ribosomes from isoprotenerol-treated hamsters. No such change was observed with ribosomes from the liver of isoproterenol-treated hamsters.

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

2016 ◽  
Vol 22 (2) ◽  
pp. 185-195 ◽  
Author(s):  
Filippo Caschera ◽  
Vincent Noireaux
Keyword(s):  
Protein Synthesis ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
Genetic Circuit ◽  
Translation System ◽  
Minimal Cell ◽  
E Coli ◽  
Synthetic Cell

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcription–translation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

scholarly journals Reconstitution of Prenyltransferase Activity on Nanodiscs by Components of the Rubber Synthesis Machinery of the Para Rubber Tree and Guayule

2021 ◽  
Author(s):  
Fu Kuroiwa ◽  
Akira Nishino ◽  
Yasuko Mandal ◽  
Masataka Honzawa ◽  
Miki Suenaga-Hiromori ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Rubber Tree ◽  
Translation System ◽  
Structure Modeling ◽  
Parthenium Argentatum ◽  
Free Translation ◽  
A Cell ◽  
Synthesis Activity ◽  
Biosynthetic Machinery ◽  
Protein Reconstitution

Abstract Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1–3) manifested prenyltransferase activity only if co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.

In search of a novel chassis material for synthetic cells: emergence of synthetic peptide compartment

Soft Matter ◽  
2020 ◽  
Vol 16 (48) ◽  
pp. 10769-10780
Author(s):  
Bineet Sharma ◽  
Yutao Ma ◽  
Andrew L. Ferguson ◽  
Allen P. Liu
Keyword(s):  
Synthetic Peptide ◽  
Cell Model ◽  
Lipid Vesicles ◽  
Synthetic Cell ◽  
Synthetic Cells

Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes. In recent years, peptide vesicles are gaining attention as an alternative chassis material.

scholarly journals Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

2021 ◽  
Author(s):  
Fu Kuroiwa ◽  
Akira Nishino ◽  
Yasuko Mandal ◽  
Miki Suenaga-Hiromori ◽  
Kakeru Suzuki ◽  
...  
Keyword(s):  
Rubber Tree ◽  
Translation System ◽  
Parthenium Argentatum ◽  
The Core ◽  
Free Translation ◽  
A Cell ◽  
Synthesis Activity ◽  
Living Organisms ◽  
Biosynthetic Machinery ◽  
Protein Reconstitution

AbstractPrenyltransferases mediate the biosynthesis of various types of polyisoprene compound in living organisms. Natural rubber (NR) of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity, with the proteins HRT1, HRT2, and HRBP having been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Cell-free synthesis of HRT1, HRT2, and HRBP in the presence of asolectin nanodiscs revealed that all three proteins were membrane associated. A complex of HRT1 and HRBP formed as a result of co-expression of the two proteins in the presence of nanodiscs manifested marked polyisoprene synthesis activity, whereas neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP exhibited such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (CPT1–3) manifested prenyltransferease activity only if co-expressed with the homolog of HRBP (CBP). Our results thus indicate that the core prenyltransferase of the rubber biosynthetic machinery of both the Para rubber tree and guayule is formed by the assembly of heterologous subunits (HRT1 and HRBP in the former species).

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