mobius assembly vector toolkit  (Addgene inc)

Journal: ACS Synthetic Biology

Article Title: A User’s Guide to Golden Gate Cloning Methods and Standards

doi: 10.1021/acssynbio.2c00355

Figure Lengend Snippet: Complete List of Golden Gate Toolkits Following the Common Syntax a

Article Snippet: Mobius Assembly Vector Toolkit , Addgene toolkit 1000000134 , ( ) , An alternative set of Level 0 backbones using AarI as assembly enzyme, and chromogenic dropouts ( amilCP , spisPink , and sfGFP ) instead of lacZα to remove the need for X-Gal and IPTG. Also, an alternative method for hierarchical assembly based on 4-part cycling (as opposed to the 2-part cycling used in GoldenBraid). , CamR , BsaI.

Techniques: Marker, Transformation Assay, Expressing, Selection, Plasmid Preparation, Infection, Clone Assay, CRISPR

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