1. 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

2. 1 Cloning & Small Scale Expression 2 Protein Expression 3 Protein Purification (Ni-NTA Affinity Column) 4 HSQC Screening 5 Analytical Gel Filtration/ Dynamic Light Scattering 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 Good Results yes no Stop work NESG Workflow Analysis Level 0 – Process Map Good Results yes No Stop Work

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

4. 1.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 Inputs Outputs SPINE/ Zebaview NESG Target List Primer Design Spreadsheet Primers Data Control Sheet Purified Gel Slice Qiagen Kit Primer Design Spreadsheet Colonies Ethanol- Precipitated DNA

5. 1.1 Procure NESG Target List Level 2 – Process Map 1.1.1 View NESG Targets 1.1.2 Execute 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 Inputs Outputs 1.2.4 Order Primers

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 Inputs Outputs 1.3.4 Manually Reconcile Target Name & well location with target size and restriction sites

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 Inputs Outputs 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

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 Inputs Outputs 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 1.5.10 Send Soluble ORFs For Sequen- cing 1.5.9 Check For Solubility

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

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

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 2.2.4C Prepare Media Inputs Outputs 2.2.3C Express In TB

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 2.4.4 Transfer Cells To 0.5 or 1.0L Inputs Outputs 2.4.3 Record Optical Density Measurements

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 2.6.4 Freeze Pellets Inputs Outputs 2.6.3 Centrifuge Tubes 2.6.5 Manually Update Fermentation Storage Database With OD Measurements

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 Inputs Outputs 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

19. 3. Protein Purification with Ni-NTA Affinity Column – Input/Output Model 3 Protein Purification With Ni-NTA Affinity Column Inputs Photograph with Information Regarding Expression level, Solubility and OD Data Pellet in -20C Freezer 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

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 3.4 Analyze Ni-NTA Elution Fractions Inputs Outputs 3.3 Run Samples Through Ni-NTA Column 3.5 Prepare Protein Samples For Screening

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 3.4 Run ExPASy Inputs Outputs 3.1.3 Add His Tag to ORF Sequence

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 Inputs Outputs 3.2.3 Centrifuge Samples

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

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 3.4.4.4 Import.gif File into SPINE Inputs Outputs 3.4.4.3 Save File as.gif

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 3.5.4A Prepare Protein Data Package Inputs Outputs 3.5.3A Exchange Buffer & Concentrate

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 3.5.4B Prepare Protein Data Package Inputs Outputs 3.5.3B Freeze Sample

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

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 Inputs Outputs 4.4 Collect 2D HSQC Data 4.5 Rate Data Qualtiy 4.6 Store HSQC Data 4.7 Update SPINS/SPINE with NMR Spectrum

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 4.1.4 Place Protein Data Package On NMR Spectroscopists Desk Inputs Outputs 4.1.3 Place NMR Samples into NMR Refrigerator

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) 4.2.4 Load NMR Sample into NMR for Screening Inputs Outputs 4.2.3 Place Sample In Clean NMR Tube

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 4.3.4 Process 1D HSQC Inputs Outputs 4.3.3 Record 1D Spectra

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

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

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

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

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 6 Preparative Gel Filtration Under Monodisperse Conditions Inputs Monodisperse Sample Conditions Entered into SPINE Outputs Protein Samples in Monodisperse Buffer Condition Gel Electrophoresis Picture of Protein Purity And Yield

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 Inputs Outputs 6.4 Analyze FPLC Results 6.5 Adjust Protein Conc. To 10 mgs/ml & Freeze 6.6 Prepare Protein Data Package

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. Inputs Outputs 6.4.4 Perform Mass Spec. 6.4.5 Perform SDS-PAGE On Pooled Fractions

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

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 Inputs Outputs 7.4 Deliver Package to Federal Express Site 7.5 Contact Crystall- ographer 7.6 Await Screening Results

43. 8. Manufacture Enriched Protein for Structural Studies – Input/Output Model 8 Manufacture Enriched Protein For Structural Studies Inputs HSQC Screening Results Crystallographic Screening Results SPINE Updated with Screening Results 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

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 Inputs Outputs 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

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 Inputs Outputs 8.1.4 Assess Collaborator Availability 8.1.5 Order Reagents

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

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 8.5.3A Repeat 3.5.2A- 3.5.4A Inputs Outputs 8.5.2A Repeat 6.4.1- 6.4.5

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 8.5.4B Prepare Protein Data Package Inputs Outputs 8.5.3B Adjust Protein Conc. To 10 mgs/ml

51. 8.6 2D HSQC NMR Spectrum of 13C,15N Level 2 – Process Map 8.6.1 Repeat 4.1, 4.2.1- 4.2.4 8.6.2 Load Previous 2D HSQC Parameters 8.6.3 Repeat 4.4.1- 4.4.3 Inputs Outputs 8.6.4 Overlay 13C,15N 2D HSQC w/ previous 2D HSQC 8.6.5 Verify Specta Are Identical

52. 9. Deliver Enriched Protein Samples for Structure Determination– Input/Output Model 9 Deliver Enriched Protein Samples For Structure Determination Inputs 1mM 13C, 15N Enriched Protein Samples In HSQC Buffer In Microtube or 10 mgs/ml SeMet Labeled Protein in Microtube Protein Data Package Outputs Federal Express Package Pickup Federal Express Tracking Number

53. 9. Deliver Enriched Protein Sample for Structure Determination Level 1 – Process Map 9.1 Repeat 7.2- 7.4 9.3 Await Further Instructions From Colloborator Inputs Outputs 9.2 Contact Collaborator With FedEx Information