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October 22, 2002 Celia Biamonte September 10,2005 Michael Baran

Workflow Analysis for the Northeast Structural Genomics Consortium at the CABM/Rutgers University/RWJMS Protein Production Facility. October 22, 2002 Celia Biamonte September 10,2005 Michael Baran. NESG Workflow Analysis Level 0 – Process Map. 1 Cloning & Small Scale Expression. 2

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October 22, 2002 Celia Biamonte September 10,2005 Michael Baran

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  1. Workflow Analysis for the Northeast Structural Genomics Consortium at the CABM/Rutgers University/RWJMSProtein Production Facility October 22, 2002 Celia Biamonte September 10,2005 Michael Baran

  2. NESG Workflow Analysis Level 0 – Process Map 1 Cloning & Small Scale Expression 2 Protein Expression 3 Protein Purification (Ni-NTA Affinity Column) 4 HSQC Screening Good Results no 5 Analytical Gel Filtration/ Dynamic Light Scattering Stop work yes 6 Preparative Gel Filtration under Monodisperse Conditions 7 Deliver Samples For Crystallographic Screening 8 Manufacture Enriched Protein for Structural Studies 9 Deliver Enriched Samples For Structure Determination yes No Stop Work Good Results

  3. 1. Cloning & Small Scale Expression – Input/Output Model • Outputs • NESG Target List • Primers • Primer Design Spreadsheet • Cloned Targets • Cloning Chart • Gel Pictures of Solubility of each ORF • Laboratory Notebook • Inputs • NESG Targets 1 Cloning & Small Scale Expression

  4. Cloning & Small Scale Expression • Level 1 – Process Map 1.1 Procure NESG Target List 1.2 Identify Targets 1.3 Perform PCR 1.4 Obtain Clone 1.5 Conduct Small- Scale Expression • SPINE/ • Zebaview • NESG Target List • Primer Design Spreadsheet • Primers • Data Control Sheet • Purified Gel Slice • Qiagen Kit • Primer Design Spreadsheet • Colonies • Ethanol-Precipitated DNA Inputs Outputs

  5. 1.1 Procure NESG Target List Level 2 – Process Map 1.1.1 View NESG Targets 1.1.2 Execute Primer Primer Program 1.1.3 Cut & Paste Results Into Excel Inputs Outputs

  6. 1.2 Identify Targets Level 2 – Process Map 1.2.1 Review NESG Target List 1.2.2 Select Organism 1.2.3 Assign Work To Analyst 1.2.4 Order Primers Inputs Outputs

  7. 1.3 Perform PCR Level 2 – Process Map 1.3.1 PCR Amplification Of ORF 1.3.2 Agarose Gel Analysis 1.3.3 ReRun PCR If Nescessary 1.3.4 Manually Reconcile Target Name & well location with target size and restriction sites Inputs Outputs

  8. 1.4 Obtain Clone Level 2 – Process Map 1.4.1 Perform Gel Extraction 1.4.2 Perform Restriction Digests 1.4.3 Purify DNA 1.4.4 Order Primers 1.4.5 Transform 1.4.6 Perform Colony PCR 1.4.7 Perfrom Mini-Prep 1.4.8 Transform into Expression Host Cells Inputs Outputs

  9. 1.5 Conduct Small Scale Expression Level 2 – Process Map 1.5.1 Select Single Colony & Grow 1.5.2 Change Media 1.5.3 Innoculate & Grow overnight 1.5.4 Innoculate Into New culture & Grow Until OD ~0.6 1.5.5 Induce 1.5.6 Harvest & Freeze Pellet 1.5.7 Sonicate 1.5.8 Perform SDS- Page 1.5.9 Check For Solubility 1.5.10 Send Soluble ORFs For Sequen- cing Inputs Outputs

  10. 2. Protein Expression – Input/Output Model • Outputs • Expressed Protein in • Cells • Gel Pictures of Protein • Expression Level, Solubility and Optical density data • Laboratory Norebook • Inputs • Plasmid • Cloning Chart • Work Order Form • From Tom Acton 2 Protein Expression

  11. 2. Protein Expression Level 1 – Process Map 2.1 Review Soluble ORFs 2.2 Select & Prepare Media 2.3 Transform Cells 2.4 Grow Cells & Measure OD 2.5 Induce Cells 2.6 Prepare & Freeze Pellets 2.7 Conduct Fermentation Analysis Inputs Outputs

  12. 2.2A Select and Prepare Media – Target Molecular Weight > 25KD Level 2 – Process Map 2.2.1A Check Molecular Weight 2.2.2A Express In TB 2.2.3A Prepare Media Inputs Outputs

  13. 2.2B Select and Prepare Media – Target Molecular Weight 15-25KD Level 2 – Process Map 2.2.1B Check Molecular Weight 2.2.2B Express In TB (1L), And MJ9 with 100% 15N (0.5L) 2.2.3B Prepare Media Inputs Outputs

  14. 2.2C Select and Prepare Media – Target Molecular Weight < 15KD Level 2 – Process Map 2.2.1C Check Molecular Weight 2.2.2C Express In MJ9 with 100% 15N & 5% 13C 2.2.3C Express In TB 2.2.4C Prepare Media Inputs Outputs

  15. 2.4 Grow Cells and Read OD Level 2 – Process Map 2.4.1 Grow 3mL of Transformed Cells in Small Tube 2.4.2 Grow Cells to 25 mL Overnight in Larger Tube 2.4.3 Record Optical Density Measurements 2.4.4 Transfer Cells To 0.5 or 1.0L Inputs Outputs

  16. 2.5 Induce Cells Level 2 – Process Map 2.5.1 Verify that Optical Density Has reached 1.0 2.5.2 Induce Cells 2.5.3 Grow Cells Overnight at 17C Inputs Outputs

  17. 2.6 Level 2 – Process Map 2.6.1 Record Optical Density 2.6.2 Take Aliquots 2.6.3 Centrifuge Tubes 2.6.4 Freeze Pellets 2.6.5 Manually Update Fermentation Storage Database With OD Measurements Inputs Outputs

  18. 2.7 Conduct Fermentation Analysis Level 2 – Process Map 2.7.1 Add Buffers To Pellets 2.7.2 Sonicate Samples 2.7.3 Centrifuge Samples 2.7.4 Perform SDS-Page 2.7.5 Take Picture & Label Gel Photo 2.7.6 Convert Image To .jpg 2.7.7 Upload .jpg To Fermentation Storage Database Inputs Outputs

  19. 3. Protein Purification with Ni-NTA Affinity Column – Input/Output Model • Outputs • Protein Samples in • Microtube • Protein Data Sheet • ExPASy Calculation • Gel Electrophoresis • Photograph with Protein • Purity, Yield, and MW • Mass Spectrometry data • Uploaded to SPINE • Inputs • Photograph • with Information • Regarding • Expression level, • Solubility and OD Data • Pellet in -20C Freezer 3 Protein Purification With Ni-NTA Affinity Column

  20. 3. Protein Purification with Ni-NTA Affinity Column Level 1 – Process Map 3.1 Calculate Molecular Weight and pI 3.2 Prepare Samples for Ni-NTA Affinity Column 3.3 Run Samples Through Ni-NTA Column 3.4 Analyze Ni-NTA Elution Fractions 3.5 Prepare Protein Samples For Screening Inputs Outputs

  21. 3.1 Calculate Molecular Weight & pI Level 2 – Process Map 3.1.1 Access ZebaView 3.1.2 Copy & Paste ORF Sequence Into ExPASy 3.1.3 Add His Tag to ORF Sequence 3.4 Run ExPASy Inputs Outputs

  22. 3.2 Prepare Samples for Ni-NTA Affinity Column Level 2 – Process Map 3.2.1 Re-suspend Pellets 3.2.2 Sonicate Samples 3.2.3 Centrifuge Samples Inputs Outputs

  23. 3.4 Analyze Ni-NTA Elution Fractions Level 2 – Process Map 3.4.1 Record OD Measurements 3.4.2 Perform SDS- PAGE 3.4.3 Pool Fractions & Determine Concentrations 3.4.4 Perform Mass Spectroscopy Inputs Outputs

  24. 3.4.4 Perform Mass Spectroscopy Level 3 – Process Map 3.4.4.1 Run Spectrum 3.4.4.2 Copy & Paste Into Excel 3.4.4.3 Save File as .gif 3.4.4.4 Import .gif File into SPINE Inputs Outputs

  25. 3.5A Prepare Protein Samples for Screening – HSQC (MJ9) Level 2 – Process Map 3.5.1A Select Buffer Based on pI 3.5.2A Calculate Amount Of Protein 3.5.3A Exchange Buffer & Concentrate 3.5.4A Prepare Protein Data Package Inputs Outputs

  26. 3.5B Prepare Protein Samples for Screening – Analytical Gel Filtration with Static / Dynamic Light Scattering Level 2 – Process Map 3.5.1B Adjust Concentration To < 3 mgs/ml 3.5.2B Add Reagents 3.5.3B Freeze Sample 3.5.4B Prepare Protein Data Package Inputs Outputs

  27. 4. Conduct HSQC Screening – Input / Output Model • Outputs • 2D HSQC Spectrum • With Priority Score • Archived NMR data • .jpg image • SPINE / SPINS updated • With NMR Spectra & Score • NMR Sample replaced in Microtube • Inputs • 15N Protein Samples • In HSQC Buffer • Protein Information • Sheet • ExPASy Calculation • Gel Picture • Mass Spec Data • NMR Request Form 4 Conduct HSQC Screening

  28. 4. Conduct HSQC Screening Level 1 – Process Map 4.1 Deposit 15N Sample Into NMR Refrigerator 4.2 Prepare Sample In NMR Tube 4.3 Collect 1D HSQC Data 4.4 Collect 2D HSQC Data 4.5 Rate Data Qualtiy 4.6 Store HSQC Data 4.7 Update SPINS/SPINE with NMR Spectrum Inputs Outputs

  29. 4.1 Deposit 15N NMR Samples into NMR Refrigerator Level 2 – Process Map 4.1.1 Contact NMR Spectroscopist For NMR Availability 4.1.2 Bring 15N NMR Samples to NMR Lab 4.1.3 Place NMR Samples into NMR Refrigerator 4.1.4 Place Protein Data Package On NMR Spectroscopists Desk Inputs Outputs

  30. 4.2 Prepare Sample in NMR Tube Level 2 – Process Map 4.2.1 Clean & Dry NMR Tube 4.2.2 Add 5% D2) (if needed) 4.2.3 Place Sample In Clean NMR Tube 4.2.4 Load NMR Sample into NMR for Screening Inputs Outputs

  31. 4.3 Collect 1D HSQC Data Level 2 – Process Map 4.3.1 Conduct initial Manual Shimming 4.3.2 Upload Standard Pulse sequence HSQC 4.3.3 Record 1D Spectra 4.3.4 Process 1D HSQC Inputs Outputs

  32. 4.4 Collect 2D HSQC Data Level 2 – Process Map 4.3.1 Conduct initial Manual Shimming 4.3.3 Record 1D Spectra 4.3.4 Process 2D HSQC Inputs Outputs

  33. 4.5 Rate Data Quality Level 2 – Process Map 4.5.1 Display Processed 2D Spectra 4.5.2 Select Score From Established Categories Inputs Outputs

  34. 4.6 Store HSQC Data Level 2 – Process Map 4.6.1 Save NMR Spectrum With Score 4.6.2 Email Biochemist Data Directory Location Inputs Outputs

  35. 4.7 Update SPINE/SPINS with NMR Spectrum Level 2 – Process Map 4.7.1 Use SPINS Interface to upload HSQC to SPINS/SPNE Inputs Outputs

  36. 5. Analytical Gel Filtration with Static/Dynamic Light Scattering – Input/Output Model • Outputs • Results uploaded to SPINE • Inputs • Concentrated Protein • Sample • with DTT, Arg, • and Glycerol • Frozen at -80C • Protein Info Sheet, • ExPASy Calc, Gel • Picture and Mass Spec • Data 5 Analytical Gel Filtration with Static/Dynamic Light Scattering

  37. 5. Analytical Gel Filtration with Static/Dynamic Light Scattering Level 1 – Process Map Inputs Outputs

  38. 6. Preparative Gel Filtration Under Monodisperse Conditions – Input/Output Model • Outputs • Protein Samples in • Monodisperse Buffer • Condition • Gel Electrophoresis • Picture of Protein Purity • And Yield • Inputs • Monodisperse • Sample • Conditions • Entered into SPINE 6 Preparative Gel Filtration Under Monodisperse Conditions

  39. 6. Conduct Preparative Gel Filtration Under Monodisperse Conditions Level 1 – Process Map 6.1 Review Monodisperse Buffer Conditions 6.2 Thaw Ni-NTA Purified Sample 6.3 Perform FPLC On Ni-NTA Purified Sample 6.4 Analyze FPLC Results 6.5 Adjust Protein Conc. To 10 mgs/ml & Freeze 6.6 Prepare Protein Data Package Inputs Outputs

  40. 6.4 Analyze FPLC Results Level 2 – Process Map 6.4.1 Record OD Results 6.4.2 Perform SDS- PAGE 6.4.3 Pool Fractions & Determine Conc. 6.4.4 Perform Mass Spec. 6.4.5 Perform SDS-PAGE On Pooled Fractions Inputs Outputs

  41. 7. Deliver Samples for Crystallographic Screening – Input/Output Model • Outputs • Concentrated Protein • Sample • Federal Express Tracking • Number • Federal Express Sample • Pickup • Inputs • Concentrated, • Frozen, • Protein Sample • Protein Information • Sheet • Gel Picture with • Protein Purity and Yield • Information • Mass Spec. Data 7 Deliver Protein Samples for Crystallographic Screening

  42. 7. Deliver Protein Samples for Crystallographic Screening Level 1 – Process Map 7.1 Concentrate & Freeze Protein Sample (10 mgs/ml) 7.2 Arrange For Federal Express Pickup 7.3 Prepare Federal Express Sample Package 7.4 Deliver Package to Federal Express Site 7.5 Contact Crystall- ographer 7.6 Await Screening Results Inputs Outputs

  43. 8. Manufacture Enriched Protein for Structural Studies – Input/Output Model • Outputs • 1mM 13C,15N Enriched • Protein Samples in HSQC • Buffer in Microtube or • 10 mgs/ml SeMet Labeled Protein in Microtube • Protein Data Package • 13C, SeMet Ordered • Inputs • HSQC Screening • Results • Crystallographic • Screening Results • SPINE Updated with • Screening Results 8 Manufacture Enriched Protein For Structural Studies

  44. 8. Manufacture Enriched Protein for Structural Studies Level 1 – Process Map 8.1 Discuss Project Priority 8.2 Conduct Small-Scale Expression 8.3 Perform Protein Expression 8.4 Execute Protein Purification With Ni-NTA Affinity column 8.5 Conduct Preparative Gel Filtration 8.6 Run 2D HSQC NMR Spectra 13C, 15N Inputs Outputs

  45. 8.1 Discuss Project Priority at Project Meeting Level 2 – Process Map 8.1.1 Check Molecular Weight Of Candidate 8.1.2 Review Screening Results 8.1.3 Check Zebaview Competition Analysis 8.1.4 Assess Collaborator Availability 8.1.5 Order Reagents Inputs Outputs

  46. 8.2 Conduct Small-Scale Expression Level 2 – Process Map 8.2.1 Pick Cone used For Initial Expression 8.2.2 Repeat 1.4.9- 1.5.9 Inputs Outputs

  47. 8.3 Perform Protein Expression Level 2 – Process Map 8.3.1 Repeat 2.2-2.7 Inputs Outputs

  48. 8.4 Execute Protein Purification with Ni-NTA Column Level 2 – Process Map 8.4.1 Repeat Steps 3.2.1 – 3.4.3 Inputs Outputs

  49. 8.5A Conduct Preparative Gel Filtration – 13C, 15N Level 2 – Process Map 8.5.1A Run FPLC in Low-Salt Buffer 8.5.2A Repeat 6.4.1- 6.4.5 8.5.3A Repeat 3.5.2A- 3.5.4A Inputs Outputs

  50. 8.5B Conduct Preparative Gel Filtration - SeMet Level 2 – Process Map 8.5.1B Run FPLC In Recommended Monodisperse Buffer 8.5.2B Repeat 6.4.1- 6.4.5 8.5.3B Adjust Protein Conc. To 10 mgs/ml 8.5.4B Prepare Protein Data Package Inputs Outputs

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