Journal: Frontiers in Bioengineering and Biotechnology

Article Title: HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale

doi: 10.3389/fbioe.2022.832176

Figure Lengend Snippet: HyperXpress workflow and effect on product formation of the most relevant optimizations. (A) The HyperXpress workflow starts with the assembly of DNA parts using LCR. This assembly includes a circularization of the DNA construct which upon addition of the components required for RCA results in an amplification of circular constructs. In order to provide optimal reaction conditions for CFPS a buffer exchange is achieved through DNA precipitation and resuspension. Upon addition of the components required for CFPS each DNA assembly is assayed for product formation. (B) Results of the sequential optimizations of the workflow compared to the corresponding 0 nM bridging oligo reaction as a negative control (Neg.). EP29: EquiPhi29™ DNA Polymerase; N2(sN)2N2: endonuclease protected random hexamer oligonucleotides; PEG prec.: in-well DNA precipitation using polyethylenglycol; res.: resuspension of the DNA precipitate in ultrapure water; p09005 coexp.: bicistronic operon plasmid with sfGFP and mKate2 genes used instead of p10024 template with monocistronic GFP reporter construct ( n = 6).

Article Snippet: Moreover, two phi-polymerases were tested to achieve a higher RCA efficiency: The wild type polymerase with a temperature optimum at 30°C (phi29 DNA Polymerase, NEB) and an engineered variant with an optimum at 40°C (EquiPhi29™, ThermoScientific).

Techniques: Construct, Amplification, Buffer Exchange, Negative Control, Random Hexamer Labeling, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Development of Rheb Depleted cells as a tool for testing relationships between Rheb localization and function. (A) Immunoblot analysis of Rheb levels and phosphorylation status of mTORC1 substrates (S6K, ULK1, and 4EBP1) in Control, Rheb siRNA-treated, Rheb Edited (Rheb hypomorph+RhebL1 KO), and Rheb Depleted (Rheb hypomorph+RhebL1 KO+Rheb siRNA) HeLa cells. (B) Quantification of phospho-S6K levels under the indicated conditions where phospho-S6K levels were divided by S6K and normalized to Control ( **P < 0.01; ** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test, n = 4). (C) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (D) Quantification of phospho-S6K levels from C. The phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection efficiency. Values were normalized to GFP-Rheb. Statistics were calculated in comparison to the GFP transfection (** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test; n = 3). (E–G) Live-cell images of GFP-Rheb, GFP-Rheb ΔCaaX , and GFP-RhebL1 in a COS-7 cells, respectively. (H) Schematic of GFP-Rheb-ER chimera that contains N-terminal GFP, Rheb ΔCaaX , and the transmembrane domain of cytochrome b5 (TMD). (I) Live-cell imaging of GFP-Rheb-ER localization. The leftmost image displays a low magnification view of GFP-Rheb-ER in a COS-7 cell. The three subsequent panels show higher magnifications of GFP-Rheb-ER and mRFP-Sec61B from the inset region. (J) Immunoblot analysis of phospho-S6K signaling in Rheb Depleted cells transfected with the indicated plasmids. (K) Quantification of phospho-S6K levels in I. The phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection. Values were normalized to GFP-Rheb. Statistics were calculated in comparison to GFP (** **P < 0.0001; ANOVA with Dunnett’s multiple comparisons test; n = 3). (L) Image showing the spatial relationship between GFP-Rheb-ER on ER tubules and LAMP1-positive lysosomes. Scale bars, 10 μm.

Article Snippet: To perform plasmid transfections, 500 ng of plasmid DNA, 1.5 μl of Fugene 6 transfection reagent (Promega, Madison, WI), and 100 μl of OptiMEM (ThermoFisher Scientific) were added to 80,000 cells per well in a six-well dish.

Techniques: Western Blot, Phospho-proteomics, Control, Transfection, Comparison, Live Cell Imaging

Journal: Molecular Biology of the Cell

Article Title: Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment

doi: 10.1091/mbc.E19-03-0146

Figure Lengend Snippet: Weak membrane interactions are optimal for Rheb-dependent mTORC1 activation. (A) Alignment of the hypervariable regions of Rheb, RhebL1, and H-Ras proteins with CaaX motifs highlighted in bold and palmitoylated cysteines in green. These cysteines were mutated to alanines (red) in the GFP-Rheb-HRas C→A mutant. (B–D) Live-cell images of GFP-Rheb, GFP-Rheb-HRas, and GFP-Rheb-HRas C→A in COS-7 cells, respectively. Scale bars, 10 µm. (E) Schematic of GFP-Rheb-H-Ras chimera that contains N-terminal GFP, Rheb ΔHVR , and the HVR of H-Ras. (F) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (G) Quantification of phospho-S6K levels from F. Phospho-S6K levels were divided by S6K and GFP values to control for loading and transfection (*** P < 0.001; **** P < 0.0001; ANOVA with Tukey’s multiple comparisons test; n = 5). (H) Schematic of myr-Rheb chimera that contains Rheb ΔCaaX and a myristoylation consensus sequence. (I) Immunoblot analysis of phospho-S6K levels in Rheb Depleted cells transfected with the indicated plasmids. (J) Quantification of phospho-S6K levels from I. Phospho-S6K levels were divided by S6K and Rheb values to control for loading and transfection (**** P < 0.0001; unpaired t test). (K) Immunoblot analysis of proteins identified in membrane and cytosolic fractions of HeLa cells. (L) Immunoblot analysis of phospho-S6K levels in HeLa cells treated with DMSO vehicle and 5 μM deltarasin for the times indicated.

Article Snippet: To perform plasmid transfections, 500 ng of plasmid DNA, 1.5 μl of Fugene 6 transfection reagent (Promega, Madison, WI), and 100 μl of OptiMEM (ThermoFisher Scientific) were added to 80,000 cells per well in a six-well dish.

Techniques: Membrane, Activation Assay, Mutagenesis, Western Blot, Transfection, Control, Sequencing