Journal: Journal of visualized experiments : JoVE

Article Title: Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

doi: 10.3791/63720

Figure Lengend Snippet: Materials

Article Snippet: Use a flow of nitrogen gas to dry the cuvette thoroughly. table ft1 table-wrap mode="anchored" t5 Name Company Catalog Number Comments 0.6 mL MCT Graduated Violet Fisher Scientific 05–408–127 6’-Trihydroxyacetophenone monohydrate 98% Sigma Aldrich 480–66–0 Acetonitrile 99.9%, HPLC grade Fisher Scientific 75–05–8 Adenosine triphosphate, 10 mM New Englang Bioscience P0756S Ammonium citrate, dibasic 98% Sigma Aldrich 3012–65–5 Ammonium Fluoride 98.0%, ACS grade Alfa Aesar 12125–01–8 Bruker bacterial test standard Bruker Daltonics 8255343 Commercial source of Xrn-1 New England BioLabs M0338S Diethyl pyrocarbonate, 97% ACROS Organics A0368487 Flex analysis software Bruker daltonics FlexAnalysis software version 3.4, Bruker Daltonics Lambda 365 UV-vis spectrophotometer Perkin Elmer MALDI plate: MSP 96 ground steel target Bruker Daltonics 280799 Mass Spectrometer Bruker Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA) Mili-Q IQ 7000 Milipore Sigma A Mili-Q system was used to purify all water used in this work Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg) Pall Corporation OD010C33 filter media, Omega (modified polyethersulfone) 10 K pore size NEBuffer 3 New England Biolabs B7003S This is solution B Oligo Analyzer tool IDT-DNA https://www.idtdna.com/calc/analyzer Pipette tips P10 Fisher Scientific 02–707–441 Pipette tips P200 Fisher Scientific 02–707–419 RNase Away Molecular BioProducts 7005–11 T4 Polynucleotide Kinase New England BioLabs M0201S T4 Polynucleotide Kinase Reaction Buffer New England BioLabs B0201S This is solution A Triflouroacetic Acid Alfa Aesar 76–05-1 Xrn-1 exoribonuclease Expressed in house See ref. 20 ZipTip Pipette Tips for Sample preparation Millipore ZTC 18S 096 10 μL pipette tips loaded with a C18 standard 0.6 μL bed Open in a separate window Materials Use a pipette to transfer the solution (step 1.1.1) into the cuvette, ensuring no air bubbles.

Techniques: Software, Spectrophotometry, Mass Spectrometry, Modification, Transferring, Sample Prep

Journal: Cell chemical biology

Article Title: Probing Exchange Units for Combining Iterative and Linear Fungal Nonribosomal Peptide Synthetases.

doi: 10.1016/j.chembiol.2019.08.005

Figure Lengend Snippet: Figure 2. Exchange of C-A(Mt)-T Units in CDP Synthetases Overview of the in vivo CDP production of wild-type and artificial synthetases in E. coli based on EnSYN (blue) and BaSYN (green) with swapped full module 2 (xM2), as observed by MALDI-TOF-MS. Hybrid units were taken from CySYN modules 2 (N-Me-L-Leu; light pink), 4 (N-Me-L-Val; pink), 7 (N-Me-Gly; dark pink), and 10 (N-Me-L-Leu; magenta). The specificities of the cognate or hybrid C2 domain at the don- and acc-sites are indicated next to each synthetase in black (intrinsically respected) and red (disrespected in the hybrid system). See also Figures S2–S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Fungal Strains E. coli DH5a (K12) Datsenko and Wanner, 2000 N/A E. coli BL21 gold (DE3)_npgA Steiniger et al., 2017 N/A Beauveria bassiana ATCC ATCC7159 Fusarium oxysporum ETH Zurich ETH 1536 Tolypocladium inflatum DSMZ DSM 915 E. coli strains containing synthetases, see Table S3 This paper N/A Chemicals, Peptides, and Recombinant Proteins Ampicillin Carl Roth Cat#K029.1 Chloramphenicol Carl Roth Cat#3886.2 D-Hydroxyisovaleric acid Dr. Stephan Gr€atz (inhouse) N/A Critical Commercial Assays CloneJET PCR Cloning Kit Thermo Fisher Cat#K1232 Oligonucleotides Primers for Synthetase cloning, see Table S1 This paper N/A Recombinant DNA Plasmids containing synthetases, see Table S2 This paper N/A Software and Algorithms Clone Manager Suite 7 Scientific & Educational Software N/A Clustal Omega Sievers et al., 2014 https://www.ebi.ac.uk/Tools/msa/clustalo/ flexAnalysis 3.4 Bruker Daltonics N/A MassHunter B.06 Agilent Technologies N/A PyMOL 1.6.0 Schrodinger, LLC https://pymol.informer.com/1.6/

Techniques: In Vivo

Journal: Cell chemical biology

Article Title: Probing Exchange Units for Combining Iterative and Linear Fungal Nonribosomal Peptide Synthetases.

doi: 10.1016/j.chembiol.2019.08.005

Figure Lengend Snippet: Figure 3. Exchange of A(Mt)-T Units in CDP Synthetases Overview of the in vivo CDP production of wild-type and artificial synthetases in E. coli based on EnSYN (blue) and BaSYN (green) with swapped A(Mt)-T part of module 2 (x(AMtT)2), as observed by MALDI-TOF-MS. Hybrid units were taken from CySYN modules 2 (N-Me-L-Leu; light pink), 4 (N-Me-L-Val; pink), 7 (N-Me-Gly; dark pink), and 10 (N-Me-L-Leu; magenta). The specificities of the cognate or hybrid C2 domain at the don- and acc-sites are indicated next to each synthetase in black (intrinsically respected), green (respected in the hybrid system), or red (disrespected). See also Figures S2–S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Fungal Strains E. coli DH5a (K12) Datsenko and Wanner, 2000 N/A E. coli BL21 gold (DE3)_npgA Steiniger et al., 2017 N/A Beauveria bassiana ATCC ATCC7159 Fusarium oxysporum ETH Zurich ETH 1536 Tolypocladium inflatum DSMZ DSM 915 E. coli strains containing synthetases, see Table S3 This paper N/A Chemicals, Peptides, and Recombinant Proteins Ampicillin Carl Roth Cat#K029.1 Chloramphenicol Carl Roth Cat#3886.2 D-Hydroxyisovaleric acid Dr. Stephan Gr€atz (inhouse) N/A Critical Commercial Assays CloneJET PCR Cloning Kit Thermo Fisher Cat#K1232 Oligonucleotides Primers for Synthetase cloning, see Table S1 This paper N/A Recombinant DNA Plasmids containing synthetases, see Table S2 This paper N/A Software and Algorithms Clone Manager Suite 7 Scientific & Educational Software N/A Clustal Omega Sievers et al., 2014 https://www.ebi.ac.uk/Tools/msa/clustalo/ flexAnalysis 3.4 Bruker Daltonics N/A MassHunter B.06 Agilent Technologies N/A PyMOL 1.6.0 Schrodinger, LLC https://pymol.informer.com/1.6/

Techniques: In Vivo

Journal: Cell chemical biology

Article Title: Probing Exchange Units for Combining Iterative and Linear Fungal Nonribosomal Peptide Synthetases.

doi: 10.1016/j.chembiol.2019.08.005

Figure Lengend Snippet: Figure 4. Exchange of CCTD-A(Mt)-T Units in CDP Synthetases Overview of the in vivo CDP production of wild-type and artificial synthetases in E. coli based on EnSYN (blue) and BaSYN (green) with swapped CCTD-A(Mt)-T part of module 2 (xCTDM2), as observed by MALDI-TOF-MS. Hybrid units were taken from CySYN module 2 (N-Me-L-Leu; light pink), 4 (N-Me-L-Val; pink), 7 (N-Me- Gly; dark pink) and 10 (N-Me-L-Leu; magenta). The specificities of the cognate or hybrid C2 domain at the don- and acc-sites are indicated next to each syn- thetase in black (intrinsically respected) or gray (might arise from EnSYN- or CySYN-derived C subdomains). See also Figures S2–S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Fungal Strains E. coli DH5a (K12) Datsenko and Wanner, 2000 N/A E. coli BL21 gold (DE3)_npgA Steiniger et al., 2017 N/A Beauveria bassiana ATCC ATCC7159 Fusarium oxysporum ETH Zurich ETH 1536 Tolypocladium inflatum DSMZ DSM 915 E. coli strains containing synthetases, see Table S3 This paper N/A Chemicals, Peptides, and Recombinant Proteins Ampicillin Carl Roth Cat#K029.1 Chloramphenicol Carl Roth Cat#3886.2 D-Hydroxyisovaleric acid Dr. Stephan Gr€atz (inhouse) N/A Critical Commercial Assays CloneJET PCR Cloning Kit Thermo Fisher Cat#K1232 Oligonucleotides Primers for Synthetase cloning, see Table S1 This paper N/A Recombinant DNA Plasmids containing synthetases, see Table S2 This paper N/A Software and Algorithms Clone Manager Suite 7 Scientific & Educational Software N/A Clustal Omega Sievers et al., 2014 https://www.ebi.ac.uk/Tools/msa/clustalo/ flexAnalysis 3.4 Bruker Daltonics N/A MassHunter B.06 Agilent Technologies N/A PyMOL 1.6.0 Schrodinger, LLC https://pymol.informer.com/1.6/

Techniques: In Vivo, Derivative Assay