Journal: PLoS ONE

Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

doi: 10.1371/journal.pone.0189892

Figure Lengend Snippet: (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

Article Snippet: The full sequences of the 16 Mobius Assembly toolkit vectors have been deposited to GenBank, and these vectors are publicly distributed from Addgene; their GenBank and Addgene IDs are listed in .

Techniques: Plasmid Preparation, Cloning, Marker, Amplification, Functional Assay, Sequencing

Journal: PLoS ONE

Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

doi: 10.1371/journal.pone.0189892

Figure Lengend Snippet: (A) Mobius Assembly uses a two-level (Level 1 and 2) approach for transcriptional unit (TU) and multi-TU augmentation. Each level is comprised of four Acceptor Vectors. The four Level 1 Acceptor Vectors (A, B, Γ, and Δ) carry spisPink gene as the visible cloning screening marker and confer Kanamycin resistance. The four Level 2 Acceptor Vectors (A, B, Γ, and Δ) carry sfGFP gene as the visible cloning screening marker and confer Chloramphenicol resistance. The standard parts stored in mUAVs are released and fused in a Level 1 reaction to form a TU. Up to four Level 1 TUs can be fused in a Level 2 reaction to form a multi-TU cassette. Switching back and forth between Level 1 and 2 leads to further expansion of multi-TUs according to the geometric sequence: 1, 4, 16, 64,…. Red arrows denote Aar I restriction sites and Purple arrows Bsa I restriction sites. (B, C, and D) E . coli colonies carrying mUAV (B), Level Acceptor 1 Vector A (C), and Level 2 Acceptor Vector A (D), which respectively exhibit purple, magenta and yellow colour after overnight incubation. Successful assembly produces white colonies.

Article Snippet: The full sequences of the 16 Mobius Assembly toolkit vectors have been deposited to GenBank, and these vectors are publicly distributed from Addgene; their GenBank and Addgene IDs are listed in .

Techniques: Cloning, Marker, Sequencing, Plasmid Preparation, Incubation

Journal: PLoS ONE

Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

doi: 10.1371/journal.pone.0189892

Figure Lengend Snippet: (A) A schematic of carotenoid biosynthetic pathway showing the enzymes mediating the production of zeaxanthin as the final product. (B) The multi-TU constructs made by Mobius Assembly to produce lycopene ( crtEBI ) and β-carotene ( crtEBIY ) (in Level 2 Acceptor Vectors) and for zeaxanthin ( crtEBIYZ ) by assembling crtEBIY and crtZ back in a Level 1 A Vector. Colonies producing lycopene are pink (C), β-carotene orange (D), and zeaxanthin yellow (E). Cells carrying intact Level 2 Vectors produced bright yellow colonies, and Level 1 Vectors pink colonies. Gel electrophoresis of the PCR from five colonies (pink, orange and yellow from each cloning, respectively) verified the correct size of the constructs; 4.3kb for lycopene (F), 5.7kb for β-carotene (G), and 6.7kb zeaxanthin (H). UV-Visible spectrophotometry showed expected peaks for lycopene (446nm, 472nm, and 503nm, I), β-carotene (450nm and 478nm, J) and zeaxanthin (450nm and 478nm, K).

Article Snippet: The full sequences of the 16 Mobius Assembly toolkit vectors have been deposited to GenBank, and these vectors are publicly distributed from Addgene; their GenBank and Addgene IDs are listed in .

Techniques: Construct, Plasmid Preparation, Produced, Nucleic Acid Electrophoresis, Cloning, Spectrophotometry

Journal: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate transforming capability and result in hyperactivation of the JAK/STAT pathway. (A) Growth curves of Ba/F3 cells expressing JAK1 A634D and JAK1 R629_S632delinsSA cultured in the absence of IL-3 (graph is representative of 3 independent experiments). (B) Immunoblot analysis of Ba/F3 parental cells, Ba/F3 cells expressing JAK1 wild-type, JAK1 A634D, and JAK1 R629_S632delinsSA cultured in the presence of IL-3 and IL-3–independent Ba/F3 cells expressing JAK1 A634D, and JAK1 R629_S632delinsSA. (C) Immunoblot analysis of HEK293 cells expressing JAK1 wild type, JAK1 A634D, and JAK1 R629_S632delinsSA. p-, phosphorylated; WT, wild-type.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Blood Advances

Article Title: A novel activating JAK1 mutation in chronic eosinophilic leukemia

doi: 10.1182/bloodadvances.2021004237

Figure Lengend Snippet: JAK1 pseudokinase mutations demonstrate sensitivity to JAK inhibitors. (A) Representative graphs of the dose-response curves (72-hour sensitivity) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (upper panels). Graphs showing mean IC50 (lower left panel) and area under the dose-response curve (AUC; lower right panel) for different JAK inhibitors on Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA (3 independent experiments). (B) Immunoblot analysis of IL-3–independent Ba/F3 cells expressing BCR-ABL, JAK1 A634D, and JAK1 R629_S632delinsSA treated with vehicle or 50 nM or 100 nM of ruxolitinib for 4 hours (n = 2 replicates). (C) Sensitivity of peripheral blood and bone marrow specimens from the patient with JAK1 R629_S632delinsSA to JAK inhibitors, represented by IC50, in comparison with control samples obtained from healthy donors (HD) and specimens from a larger cohort of patients with MPNs) (the horizontal lines denote median IC50 of HD and MPN specimens). ****P < .0001, ***P < .001, 1-way analysis of variance. p-, phosphorylated.

Article Snippet: Vectors, cloning, cell culture, retrovirus generation, Ba/F3 transformation assays, measurement of drug response by cell proliferation assay, and immunoblotting JAK1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002227.2","term_id":"102469033","term_text":"NM_002227.2"}} NM_002227.2 ) variant c.1887_1894delinsTG, p.Arg629_Ser632delinsSerAla, and c.1901C>A, p.A634D pDONR vectors were generated in pDONR223-JAK1 vector (Addgene plasmid #23932).

Techniques: Expressing, Western Blot, Comparison, Control

Journal: Frontiers in Plant Science

Article Title: Biocircuits in plants and eukaryotic algae

doi: 10.3389/fpls.2022.982959

Figure Lengend Snippet: Synthetic biology tools for plants and algae.

Article Snippet: , , MoClo CRISPR/Cas toolkit for Plants ( https://www.addgene.org/kits/nekrasov-moclo-plant-crispr/ ) , Kit collection with 95 plasmids for transformation and expression of CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters in plants. , .

Techniques: Algae, Transformation Assay, Construct, Translocation Assay, CRISPR, Expressing, Functional Assay, Sequencing, In Silico, Software, Bacteria, Gene Expression