Journal: Plants

Article Title: The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis

doi: 10.3390/plants8110516

Figure Lengend Snippet: Summary of the tools available for polysaccharide production in different host organisms. For each species, a relative ranking for different attributes is shown: handling, from least (+) to most convenient (++++); and price, from cheap ($) to most expensive ($$$$). The relative number of strains and vectors available to order were checked at AddGene ( www.addgene.org ), ATCC ( www.atcc.org ), DSMZ ( www.dsmz.de ), Invitrogen, and Sigma-Aldrich. Cultivation costs were based on the complexity of media used, the relative price of components at Sigma-Aldrich, and for the type of incubator used. Doubling times (20 min to 24 h) were estimated based on work by Pichia [ 30 ], Yarrowia [ 31 ], Ustilago [ 32 ], Pseudomonas [ 33 ], Bacillus [ 34 ], Synechocystis [ 35 ], Synechococcus [ 36 ], and DSMZ (other species). The knockout libraries for different hosts have been generated and are ready to order from AddGene, Dharmacon or AcceGen. The asterisk (*) indicates that the HEK293 mutant collection is not as comprehensive as the rest. “-“ – not described; E.coli—Escherichia coli DB3.1; Pseudomonas fluorescens ; Bacillus subtilis ; Synechocystis PCC 6803: Synechococcus elongatus sp. PCC 7942; Saccharomyces cerevisiae ; Pichia pastoris ; Yarrowia lipolytica ; Ustilago maydis; HEK293 – Human embryonic kidney cells 293; CHO—Chinese hamster ovary cells K1. CWGT—Cell wall-related glycosyltransferase; PTMs—post-translational modifications.

Article Snippet: Several collections of versatile vectors are now available from the AddGene repository for Pichia (GoldenPiCS [ ] and MoClo Pichia toolkit [ ]), and Yarrowia lipolytica (YaliBricks [ ], GoldenMOCS [ ], and EasyCloneYALI [ ]) featuring constitutive and inducible promoters, a variety of transcriptional terminators, and selection markers.

Techniques: Knock-Out, Generated, Mutagenesis, Bacteria

Journal: Plants

Article Title: The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis

doi: 10.3390/plants8110516

Figure Lengend Snippet: Glycosidic linkage analysis of isolated extracellular matrices from various organisms. Organisms were grown as described in Supplemental Materials and Methods and used to determine the composition of the alcohol-insoluble residue (AIR). Shown is the percentage of the total ion chromatogram peak area of the corresponding partially methylated, acetylated alditol representing the linked sugar. Highlighted in blue are glycosyl-moieties that also represent a glycosyl-linkage present in a plant polysaccharide: 3-Glc—mixed-linkage glucan, callose; 4-Glc—starch, cellulose, xyloglucan, glucomannan; 4,6-Glc—starch, cellulose, xyloglucan; 4-Man, 4,6-Man, t-Gal—heteromannan; 4-Xyl—xylan; 2-Rhap, 2,4-Rhap—RG-I. Data show mean ± standard deviation of three biological replicates. “Empty cell”—not detected; a—exact sugar moiety unknown; E. coli — Escherichia coli DB3.1; Pseudomonas fluorescens WCS417r; Bacillus subtilis ; Synechocystis PCC 6803; Synechococcus elongatus sp. PCC 7942; Saccharomyces cerevisiae BY4742; Pichia pastoris X-33; Yarrowia lipolytica Po1d; Ustilago maydis AB31; CHO—Chinese hamster ovary cells K1.

Article Snippet: Several collections of versatile vectors are now available from the AddGene repository for Pichia (GoldenPiCS [ ] and MoClo Pichia toolkit [ ]), and Yarrowia lipolytica (YaliBricks [ ], GoldenMOCS [ ], and EasyCloneYALI [ ]) featuring constitutive and inducible promoters, a variety of transcriptional terminators, and selection markers.

Techniques: Isolation, Residue, Methylation, Standard Deviation

Journal: Plants

Article Title: The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis

doi: 10.3390/plants8110516

Figure Lengend Snippet: Comparison of fluorescent protein expression in two different yeast species. Pichia (blue) and Yarrowia (orange) cells after 24 h of growth in methanol- or erythritol-containing media (see ), respectively, and expressing no transgene (empty vector) or sfGFP under control of the methanol-inducible pAOX1 , the constitutive pTEF , or the erythritol-inducible promoter pEYK . ( A ) Widefield micrographs of transmitted light (TL) and GFP signals in yeast cells after exposure (time indicated in each panel) to a halogen light source. Scale bars = 10 µm. ( B ) Relative fluorescence units (RFU; normalized to optical density at 600 nm) of yeast cells quantified with a plate reader. Different letters indicate significant changes based on one-way ANOVA with post hoc Tukey HSD Test ( P < 0.05). Data show mean ± SD of three biological replicates.

Article Snippet: Several collections of versatile vectors are now available from the AddGene repository for Pichia (GoldenPiCS [ ] and MoClo Pichia toolkit [ ]), and Yarrowia lipolytica (YaliBricks [ ], GoldenMOCS [ ], and EasyCloneYALI [ ]) featuring constitutive and inducible promoters, a variety of transcriptional terminators, and selection markers.

Techniques: Comparison, Expressing, Plasmid Preparation, Control, Fluorescence

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: MoClo CRISPR/Cas Toolkit for Plants , Addgene toolkit 1000000159 , ( ) , CRISPR/Cas nucleases and gRNA backbones. , SpeR or KanR , BsaI.

Techniques: Marker, Transformation Assay, Expressing, Ubiquitin Proteomics, Selection, Plasmid Preparation, Infection, Cloning, CRISPR, Bacteria

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: Alternative Golden Gate Methods That Do Not Follow the Common Syntax a

Article Snippet: MoClo CRISPR/Cas Toolkit for Plants , Addgene toolkit 1000000159 , ( ) , CRISPR/Cas nucleases and gRNA backbones. , SpeR or KanR , BsaI.

Techniques: Selection, Expressing, Marker

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