Journal: Cell

Article Title: Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome

doi: 10.1016/j.cell.2022.03.008

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals Chloramphenicol Sigma C0378 Spectinomycin DOT Scientific DSS23000–5 Kanamycin American Bio AB01100 Apramycin Fisher AAJ66616 Carbenicillin Sigma C1389 G418 Fisher 10131035 Hygromycin B Fisher 10687010 Luria Broth American Bio AB01198 M9 Minimal Media Fisher DF0485–17 Erythromycin Acros 227330050 Amberlite XAD-7 Resin Acros 202245000 Celite Acros 349675000 Critical Commercial Assays Luna Universal qRT-PCR kit NEB E3005 Purexpress translation kit NEB E6800S HiScribe T7 RNA Kit NEB E2040S Experimental Models: Organisms and Strains See Extended Data S2 for detailed list Indicated when externally acquired Recombinant DNA Source of PhiC31 Integrase (Groth et al., 2000) Addgene 18941 Source of Tn5 Transposase ( Martínez-García et al., 2011 ) Addgene 61564 Source of miniR6K origin (Puri et al., 2015) Addgene 61263 Source of pAMβ1 origin (O’Sullivan and Klaenhammer, 1993) Addgene 71312 See Data S2 and Figure S4 , for detailed description of all additional constructs designed for this study Software and Algorithms ChemDraw 20 PerkinElmer https://perkinelmerinformatics.com/products/research/chemdraw/ Mnova Mestrelab Research https://mestrelab.com/download/mnova/ Prism 7 Graphpad https://www.graphpad.com/ Adobe Illustrator CC Adobe Adobe.com Python 3 https://www.python.org/ Transtermhp 2.08 ( Kingsford et al., 2007 ) https://transterm.cbcb.umd.edu Vienna RNA Suite 2.4.14 ( Lorenz et al., 2011 ) https://www.tbi.univie.ac.at/RNA/ NuPoP 3 ( Xi et al., 2010 ) https://bioconductor.org/packages/release/bioc/html/NuPoP.html R 4 https://www.r-project.org/ phyloT v2 https://phylot.biobyte.de/ iTOL v6 (Letunic and Bork, 2021) https://itol.embl.de/ DNAplotlib (Der et al., 2017) https://github.com/VoigtLab/dnaplotlib cBlaster ( Gilchrist et al., 2021 ) https://cblaster.readthedocs.io/en/latest/index.html Open in a separate window Deposited Data: https://data.mendeley.com/datasets/pzbz8yjfz6/draft?a=d468c163-bb50–4611-a10c-278e8afa0a98.

Techniques: Recombinant, Construct, Software

Journal: Cell

Article Title: Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome

doi: 10.1016/j.cell.2022.03.008

Figure Lengend Snippet: (A) Coupled conjugation-transposition is used to deliver a landing pad into a recipient genome. pX: “seeding” promoter, either pIP433 P1 or absent. (B) Genetic cargo is integrated via a phiC31 integrase at the cognate attP sites. (C) ~2000 clones containing the landing pad in E. coli MG1655 were pooled and assayed by flow cytometry. Fluorescence distribution of uninduced (pink) and induced (green, theophylline + aTc) cells was quantified. Four individual clones were randomly picked and similarly quantified with and without induction. Mean and coefficient of variation (CV) are shown to quantify expression strength and variability, respectively. (D) The landing pad was introduced into various bacterial strains, and GFP induction was quantified for 7 randomly-selected transconjugant clones. Fluorescence is collected on a plate reader 12 hours after theophylline and aTc induction. aTc is constant (100 ng/mL) in all induced conditions. (E) The violacein biosynthetic pathway was cloned with its native sequence under its native promoter element, under the orthogonal T7 promoter, and as a fully redesigned SGE. (F) Production of violacein was quantified through absorbance in its native host C. violaceum and in landing pad-domesticated P. putida. Production of violet pigment was quantified by absorbance at 585 nm; cell density was quantified by absorbance at 660 nm. P. putida strains were induced with 1 mM theophylline + 100 ng/mL aTc. Data were analyzed with a 2-way t-test. *P < 0.05; ***P < 0.001. (G) Production and quantification of violacein, and strain fitness is performed in other landing pad-domesticated bacterial hosts. [Theophylline] is varied while aTc was fixed at 100 ng/mL in all induced conditions.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals Chloramphenicol Sigma C0378 Spectinomycin DOT Scientific DSS23000–5 Kanamycin American Bio AB01100 Apramycin Fisher AAJ66616 Carbenicillin Sigma C1389 G418 Fisher 10131035 Hygromycin B Fisher 10687010 Luria Broth American Bio AB01198 M9 Minimal Media Fisher DF0485–17 Erythromycin Acros 227330050 Amberlite XAD-7 Resin Acros 202245000 Celite Acros 349675000 Critical Commercial Assays Luna Universal qRT-PCR kit NEB E3005 Purexpress translation kit NEB E6800S HiScribe T7 RNA Kit NEB E2040S Experimental Models: Organisms and Strains See Extended Data S2 for detailed list Indicated when externally acquired Recombinant DNA Source of PhiC31 Integrase (Groth et al., 2000) Addgene 18941 Source of Tn5 Transposase ( Martínez-García et al., 2011 ) Addgene 61564 Source of miniR6K origin (Puri et al., 2015) Addgene 61263 Source of pAMβ1 origin (O’Sullivan and Klaenhammer, 1993) Addgene 71312 See Data S2 and Figure S4 , for detailed description of all additional constructs designed for this study Software and Algorithms ChemDraw 20 PerkinElmer https://perkinelmerinformatics.com/products/research/chemdraw/ Mnova Mestrelab Research https://mestrelab.com/download/mnova/ Prism 7 Graphpad https://www.graphpad.com/ Adobe Illustrator CC Adobe Adobe.com Python 3 https://www.python.org/ Transtermhp 2.08 ( Kingsford et al., 2007 ) https://transterm.cbcb.umd.edu Vienna RNA Suite 2.4.14 ( Lorenz et al., 2011 ) https://www.tbi.univie.ac.at/RNA/ NuPoP 3 ( Xi et al., 2010 ) https://bioconductor.org/packages/release/bioc/html/NuPoP.html R 4 https://www.r-project.org/ phyloT v2 https://phylot.biobyte.de/ iTOL v6 (Letunic and Bork, 2021) https://itol.embl.de/ DNAplotlib (Der et al., 2017) https://github.com/VoigtLab/dnaplotlib cBlaster ( Gilchrist et al., 2021 ) https://cblaster.readthedocs.io/en/latest/index.html Open in a separate window Deposited Data: https://data.mendeley.com/datasets/pzbz8yjfz6/draft?a=d468c163-bb50–4611-a10c-278e8afa0a98.

Techniques: Conjugation Assay, Clone Assay, Flow Cytometry, Fluorescence, Expressing, Sequencing

Journal: Cell

Article Title: Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome

doi: 10.1016/j.cell.2022.03.008

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals Chloramphenicol Sigma C0378 Spectinomycin DOT Scientific DSS23000–5 Kanamycin American Bio AB01100 Apramycin Fisher AAJ66616 Carbenicillin Sigma C1389 G418 Fisher 10131035 Hygromycin B Fisher 10687010 Luria Broth American Bio AB01198 M9 Minimal Media Fisher DF0485–17 Erythromycin Acros 227330050 Amberlite XAD-7 Resin Acros 202245000 Celite Acros 349675000 Critical Commercial Assays Luna Universal qRT-PCR kit NEB E3005 Purexpress translation kit NEB E6800S HiScribe T7 RNA Kit NEB E2040S Experimental Models: Organisms and Strains See Extended Data S2 for detailed list Indicated when externally acquired Recombinant DNA Source of PhiC31 Integrase (Groth et al., 2000) Addgene 18941 Source of Tn5 Transposase ( Martínez-García et al., 2011 ) Addgene 61564 Source of miniR6K origin (Puri et al., 2015) Addgene 61263 Source of pAMβ1 origin (O’Sullivan and Klaenhammer, 1993) Addgene 71312 See Data S2 and Figure S4 , for detailed description of all additional constructs designed for this study Software and Algorithms ChemDraw 20 PerkinElmer https://perkinelmerinformatics.com/products/research/chemdraw/ Mnova Mestrelab Research https://mestrelab.com/download/mnova/ Prism 7 Graphpad https://www.graphpad.com/ Adobe Illustrator CC Adobe Adobe.com Python 3 https://www.python.org/ Transtermhp 2.08 ( Kingsford et al., 2007 ) https://transterm.cbcb.umd.edu Vienna RNA Suite 2.4.14 ( Lorenz et al., 2011 ) https://www.tbi.univie.ac.at/RNA/ NuPoP 3 ( Xi et al., 2010 ) https://bioconductor.org/packages/release/bioc/html/NuPoP.html R 4 https://www.r-project.org/ phyloT v2 https://phylot.biobyte.de/ iTOL v6 (Letunic and Bork, 2021) https://itol.embl.de/ DNAplotlib (Der et al., 2017) https://github.com/VoigtLab/dnaplotlib cBlaster ( Gilchrist et al., 2021 ) https://cblaster.readthedocs.io/en/latest/index.html Open in a separate window Deposited Data: https://data.mendeley.com/datasets/pzbz8yjfz6/draft?a=d468c163-bb50–4611-a10c-278e8afa0a98.

Techniques: Recombinant, Construct, Software

Journal: Cell

Article Title: Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome

doi: 10.1016/j.cell.2022.03.008

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals Chloramphenicol Sigma C0378 Spectinomycin DOT Scientific DSS23000–5 Kanamycin American Bio AB01100 Apramycin Fisher AAJ66616 Carbenicillin Sigma C1389 G418 Fisher 10131035 Hygromycin B Fisher 10687010 Luria Broth American Bio AB01198 M9 Minimal Media Fisher DF0485–17 Erythromycin Acros 227330050 Amberlite XAD-7 Resin Acros 202245000 Celite Acros 349675000 Critical Commercial Assays Luna Universal qRT-PCR kit NEB E3005 Purexpress translation kit NEB E6800S HiScribe T7 RNA Kit NEB E2040S Experimental Models: Organisms and Strains See Extended Data S2 for detailed list Indicated when externally acquired Recombinant DNA Source of PhiC31 Integrase (Groth et al., 2000) Addgene 18941 Source of Tn5 Transposase ( Martínez-García et al., 2011 ) Addgene 61564 Source of miniR6K origin (Puri et al., 2015) Addgene 61263 Source of pAMβ1 origin (O’Sullivan and Klaenhammer, 1993) Addgene 71312 See Data S2 and Figure S4 , for detailed description of all additional constructs designed for this study Software and Algorithms ChemDraw 20 PerkinElmer https://perkinelmerinformatics.com/products/research/chemdraw/ Mnova Mestrelab Research https://mestrelab.com/download/mnova/ Prism 7 Graphpad https://www.graphpad.com/ Adobe Illustrator CC Adobe Adobe.com Python 3 https://www.python.org/ Transtermhp 2.08 ( Kingsford et al., 2007 ) https://transterm.cbcb.umd.edu Vienna RNA Suite 2.4.14 ( Lorenz et al., 2011 ) https://www.tbi.univie.ac.at/RNA/ NuPoP 3 ( Xi et al., 2010 ) https://bioconductor.org/packages/release/bioc/html/NuPoP.html R 4 https://www.r-project.org/ phyloT v2 https://phylot.biobyte.de/ iTOL v6 (Letunic and Bork, 2021) https://itol.embl.de/ DNAplotlib (Der et al., 2017) https://github.com/VoigtLab/dnaplotlib cBlaster ( Gilchrist et al., 2021 ) https://cblaster.readthedocs.io/en/latest/index.html Open in a separate window Deposited Data: https://data.mendeley.com/datasets/pzbz8yjfz6/draft?a=d468c163-bb50–4611-a10c-278e8afa0a98.

Techniques: Recombinant, Construct, Software