Journal: Nature Communications

Article Title: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

doi: 10.1038/s41467-018-03926-1

Figure Lengend Snippet: Basic elements of the DNA self-replication strategy. a Flow of genetic information reconstituted in batch mode reaction or inside a liposome. The Φ29 virus-inspired DNA replication mechanism is implemented in the PURE frex transcription-translation system. A DNA replication cycle is completed when the DNA template expressing the Φ29 proteins is also the replicating DNA. Alternatively, the expressing DNA does not contain the Φ29 origin sequences (oriLR- in brackets) and a different target DNA is used as a replication substrate (solid colored line). Some essential reaction substrates are indicated. b Schematic depicting the mechanism of replication initiation by the Φ29 system. c Schematic of the Φ29 genome and four de novo designed DNA constructs used in this study (Supplementary Table ). The most relevant regulatory elements are depicted: the T7 promoter (arrows), the vesicular stomatitis virus (VSV) internal terminator or the T7 terminator at the 3′ end (T, for both terminators), the genes (rectangles) and the Φ29 origins of replication. Their termination efficiency was experimentally estimated (Supplementary Fig. ). d , e Analysis of translation products on polyacrylamide protein gels. PURE frex solution was supplemented with BODIPY-Lys-tRNA Lys (GreenLys) to introduce fluorescent lysine residues in the synthesized proteins and distinguish them from the IVTT protein background ( e ). Coomassie Blue staining was also performed to visualize the purified proteins and the total protein content in PURE frex reactions ( d ). Amounts of purified proteins: 180 ng p2, 2 µg p5, 180 ng p3, and 2 µg p6. The estimated concentrations are 1.0 µM for DNAP, 4.0 µM for TP, 5.0 µM for p5 and 1.7 µM for p6, when all genes are separately expressed (Supplementary Note and Supplementary Fig. ). The production of all four full-length proteins was confirmed when the oriLR-p2-p3 and oriLR-p6-p5 templates were co-expressed in equimolar amounts (1:1) or with an excess of the oriLR-p6-p5 DNA (∼1:13), the latter ratio being used in replication experiments, where larger amounts of p5 and p6 are required. Note also the generation of truncated translation products, in particular for p2 and p3. Predicted molecular masses are 12 kDa for p6, 13.3 kDa for p5, 31 kDa for p3, and 66 kDa for p2

Article Snippet: The cell-free gene expression system PURE frex was purchased from GeneFrontier Corporation (Japan).

Techniques: Virus, Expressing, Construct, Introduce, Synthesized, Staining, Purification

Journal: Nature Communications

Article Title: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

doi: 10.1038/s41467-018-03926-1

Figure Lengend Snippet: Replication of DNA by its encoded proteins. a IVTTR reaction scheme using the oriLR-p2-p3 DNA template. Short amplification products are not represented. b The replication products of either the oriLR-p2-p3 or the p2-p3 DNA template (100 ng input) expressed in PURE frex were visualized on agarose gel after RNase and Proteinase K treatments, followed by RNeasy clean-up column purification. The results from five independent replication experiments are shown in Supplementary Fig. , Supplementary Fig. and Supplementary Fig. . In each IVTTR reaction triggered by the expression of the oriLR-p2-p3 DNA construct, 2.5 nM of template produced about 100 nM of p2 and 700 nM of p3 proteins (as estimated in Supplementary Fig. ), which were able to generate ~50 nM of full-length DNA product when the reaction was supplemented with purified p5 and p6. c Samples were further incubated with λ-exonuclease to remove TP-uncapped DNA. The asterisk indicates full-length TP-capped DNA that has not been degraded by the λ-exonuclease. d De novo synthesized DNA was subsequently used as a template for a second IVTT reaction. The translation products were visualized by PAGE with GreenLys labeling. Expression of DNA that resulted from an IVTTR in the presence of purified p5 and p6 proteins led to fluorescent p2 and p3 protein bands of similar intensity as that measured when starting with 2.5 nM purified DNA (control with PCR product) demonstrating that the encoded functions are retained during amplification. Protein gels from two independent replication experiments are shown in Supplementary Fig. and Supplementary Fig. . Note that the modest replication efficiency in the absence of purified p5 and p6 was sufficient to generate the encoded p2 and p3 proteins through amplification of information at the transcription and, to a lower extent, at the translation levels

Article Snippet: The cell-free gene expression system PURE frex was purchased from GeneFrontier Corporation (Japan).

Techniques: Amplification, Agarose Gel Electrophoresis, Purification, Expressing, Construct, Produced, Incubation, Synthesized, Labeling, Control

Journal: Nature Communications

Article Title: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

doi: 10.1038/s41467-018-03926-1

Figure Lengend Snippet: Potentiating DNA self-replication with 5′-end pre-bound TP. a IVTTR reaction scheme using the TP- oriLR-p2-p3 DNA template. Short amplification products are not represented. The detailed experimental workflow, including preparation of the TP- oriLR-p2-p3 DNA, is shown in Supplementary Fig. . b The replication products of the TP- oriLR-p2-p3 DNA template (∼75 ng input, equiv. ∼1.9 nM) expressed in PURE frex were visualized on agarose gel after RNase and Proteinase K treatments, followed by RNeasy clean-up column purification. When indicated the p6-p5 DNA (70 ng input, equiv. ∼5.7 nM) was co-expressed. The results from two independent IVTTR experiments are shown in Supplementary Fig. 12c, f . For direct comparison of the amplification yield with and without parental TP, similar amounts of input DNA were used, the end-point reaction solutions were loaded on the same gel and the band intensities were analysed (Supplementary Fig. ). Clearly, replication of the TP- oriLR-p2-p3 DNA template is more efficient. c Samples were further incubated with λ-exonuclease to remove TP-uncapped DNA. Note that the overall amount of DNA on the gel is reduced (to the extent that the band corresponding to the input TP- oriLR-p2-p3 DNA in the –dNTPs control sample is no longer visible) after nuclease treatment due to dilution during the cleaning/purification steps. d De novo synthesized DNA was subsequently used as a template for a third IVTT reaction. The translation products were visualized by PAGE with GreenLys labeling. The protein gel analysis from an independent IVTTR experiment is shown in Supplementary Fig. . e Autocatalytic IVTTR cycles realized in this study. A first IVTTR reaction was performed using oriLR-p2-p3 as input DNA and producing larger amount of TP- oriLR-p2-p3 (Supplementary Fig. 12b, e ). The purified TP- oriLR-p2-p3 DNA was subsequently used as template for a second IVTTR ( b ). Finally, the purified DNA products from IVTTR 2 was used for a third IVTT ( d )

Article Snippet: The cell-free gene expression system PURE frex was purchased from GeneFrontier Corporation (Japan).

Techniques: Amplification, Agarose Gel Electrophoresis, Purification, Comparison, Incubation, Control, Synthesized, Labeling

Journal: Nature Communications

Article Title: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

doi: 10.1038/s41467-018-03926-1

Figure Lengend Snippet: Compartmentalization of self-encoded DNA replication inside liposomes. Images in a and b display fluorescence confocal micrographs of PURE frex -containing phospholipid vesicles labeled with a membrane dye (red). a Fluorescence emitted by the YFP synthesized from its gene (7.4 nM bulk concentration of dsDNA) is visualized in green (overlaid channels). Assuming that the entrapped DNA molecules follow a Poisson distribution, liposomes with a diameter of 4 µm contain ~140 DNA copies on average. Thousands of gene-expressing liposomes can be imaged per sample. About 30% of the liposomes produce YFP at a detectable level. This functional heterogeneity is probably a consequence of the compositional diversity of the biochemical network within vesicles. Scale bar is 20 µm. b Following the IVTTR reaction scheme shown in Fig. , 5 nM of the oriLR-p2-p3 DNA template along with the purified p5 and p6 proteins were co-encapsulated with or without dNTPs during liposome formation. After gene expression and liposome immobilization, the DNA staining fluorophore acridine orange (green channel) was injected. Amplification of DNA in the lumen of individual vesicles is accompanied by a higher fluorescence signal of acridine orange in the form of bright spots. We noticed that acridine orange can stain the liposome membrane, presumably due to the hydrophobic nature of its aromatic groups. Nonetheless, the DNA and membrane signals can easily be discriminated by using the red membrane dye for co-localization analysis, so that the lumen signal from amplified DNA can unambiguously be ascribed. The fluorescence images show representative fields of view from three independent experiments. Five different fields of view of similar liposome density were analysed per experiment to quantify the number of ‘nucleoids’. Comparing + dNTPs and –dNTPs (+/–) in the three experimental repeats, 350/9, 130/2, and 773/58 nucleoid-like structures were identified. Scale bars represent 20 µm. c Line intensity profiles from eight liposomes framed in b . In the images from the –dNTPs sample, we deliberately chose liposomes exhibiting green spots to show that they co-localize with the membrane dye, demonstrating that they are of different nature than those triggered by DNA replication. Color coding is the same as in b . a.u., arbitrary units

Article Snippet: The cell-free gene expression system PURE frex was purchased from GeneFrontier Corporation (Japan).

Techniques: Liposomes, Fluorescence, Labeling, Membrane, Synthesized, Concentration Assay, Expressing, Functional Assay, Purification, Gene Expression, Staining, Injection, Amplification

Journal: Nature Communications

Article Title: Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

doi: 10.1038/s41467-018-03926-1

Figure Lengend Snippet: A semisynthetic cell with implemented Φ29-based linear DNA replication. A prospective minimal cell, whose chassis is based on the PURE frex protein factory encapsulated inside phospholipid vesicles, is represented with its essential functional modules. The transfer of information (black solid lines) from DNA to protein is executed by the PURE system. Dashed lines indicate catalysis reactions. The DNA replication module, whereby the linear genomic DNA is capped with the Φ29 TP protein (triangles) and is replicated by the Φ29 DNA synthesis machinery, has been implemented in this study (purple). Other subsystems include the regeneration of all PURE system components from their genes, the synthesis of phospholipids for the growth of the compartment, the expression of division proteins, and incorporation of transmembrane proteins (channels, transporters) to regulate the molecular diffusion with the external environment, in particular of energy-rich compounds. One challenge to realize a fully functional cell will be to efficiently interface and coordinate the different modules, something that could be fostered by DNA replication through random generation and in vitro selection of favorable phenotypic traits. Gene regulatory circuits (not depicted) could be implemented to orchestrate the expression dynamics of the different modules

Article Snippet: The cell-free gene expression system PURE frex was purchased from GeneFrontier Corporation (Japan).

Techniques: Functional Assay, DNA Synthesis, Expressing, Diffusion-based Assay, In Vitro, Selection

Journal: STAR Protocols

Article Title: Protocol for in vitro phospholipid synthesis combining fatty acid synthesis and cell-free gene expression

doi: 10.1016/j.xpro.2024.103051

Figure Lengend Snippet:

Article Snippet: Cell-free systems are commercially available as PURE frex from GeneFrontier, or as PURExpress from New England Biolabs.

Techniques: Recombinant, Marker, Staining, Software, Blocking Assay, Membrane, Electrophoresis

Journal: American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics

Article Title: Association analysis of the PIP4K2A gene on chromosome 10p12 and schizophrenia in the Irish study of high density schizophrenia families (ISHDSF) and the Irish case-control study of schizophrenia (ICCSS)

doi: 10.1002/ajmg.b.30982

Figure Lengend Snippet: Association Analysis of PIP4K2A SNPs in the FH− Cases Compared to Controls

Article Snippet: Only rs1417374 ( P = 0.004) and haplotype rs1417374-rs1409395 ( P = 0.0008) remained significant after 100,000 permutations (perm P = 0.023, perm P = 0.004 respectively). table ft1 table-wrap mode="anchored" t5 caption a7 SNP Case freq Control freq Chi-sq P Perm P Odds ratio rs1417374 0.343 0.282 8.54 0.004 0.024 1.22 rs1409395 0.367 0.317 5.43 0.020 0.158 1.16 rs10828317 0.351 0.322 1.97 0.161 0.807 1.09 rs746203 0.414 0.368 4.68 0.031 0.230 1.13 rs8341 0.401 0.355 4.51 0.034 0.247 1.13 rs1417374-rs1409395 Haplotype 1-1 0.54 0.59 8.869 0.0008 0.004 0.88 1–2 0.11 0.09 1.497 0.033 0.590 1.20 2-1 0.14 0.13 1.413 0.366 0.985 1.10 2-2 0.22 0.19 3.019 0.032 0.242 1.24 Open in a separate window Significant (uncorrected) P -values are in bold.

Techniques: Control