Implementation of High Through Put DNA Production Pipeline

Transcription

1 1 Implementation of High Through Put DNA Production Pipeline Karen Billeci PhyNexus User Meeting 8/27/2014

2 Overview 2 Biologics Resource Management Background Infrastructure Implementation of Automation to support DNA productions Comments on sample management Transformation Colony Picking Purification

3 GNE expression Constructs Biologics Resources Management 3 Mission: Provide state of the art nucleic acid resource and protein repository management to support pathway discovery, antibody discovery, and protein chemistry. Responsibilities: Implement automation, logics strategies and bioinformatics to support Biologics Resource Management. RNAi Manage Vendor Libraries and Collections to support Genentech research. Preserve Genentech resources through the archive and management of constructs and proteins. Support therapeutic discovery by providing sample management support to meet business needs. Manage DNA production for protein expression and screening applications. Vendor DNA Collections Collections

4 Biologics Resources: Infrastructure Biologics Resource Management relies on integration of Registration, Inventory Management, Automation, Request Systems, Collaboration, and logistics. Core Infrastructure 4 Request Protein Cloning Antibodies Sample Management and Archive Synthesis Inventory Management LIMS Registration System Automation Distribution CMOs Expression / Purification Assay (screening, sequencing)

5 Overview 5 Biologics Resource Management Background Infrastructure Implementation of Automation to support DNA productions Comments on sample Management Transformation Colony Picking Purification

6 Why Automate? Small Scale DNA Production Metrics Mini Scale (10 ug) 22-24,000 Preps/Month Midi Scale (50 ug) Nearing 600 Preps/Month 6

7 DNA Production Management Plasmid based reagents, cdna, shrna, and DNA constructs, are perpetually regrown and expanded starting from glycerol stocks or purified DNA. Modular Automation required to allow flexibility, while maintaining through put. Glycerol Stock Purified DNA Transformation Protein Production Pick colony Purify DNA Culture Screen

8 sirna Automation and Protein Hamilton Starlet Mini Oasis Bravo Tecan Cherry Pick Transform Inoculate Colony Picking Barcode to Barcode Tracking Hamilton Star Hudson Inventory Management LIMS Repository Fetch/Location Management Mini Purification (16-20X96 well Blocks) Dynamic Devices / PhyNexus -80 C Future 4 C -20 C Inventory Management LIMS-uses orders to define work and send/retrieve information from automation.

9 Percent of T0 Sample Management Automation Implementation Boutique sample management no longer possible. Glycerol Stocks stored at -80 C or under nitrogen Cultures traditionally from glycerol stocks started with stabs. Difficult in a HT environment 9 Glycerol Stock ARE stable to Freeze Thaw 40% High throughput sample management Store general use collections as glycerol stocks at (-80 C) and purified DNA (-20 C) Glycerol stocks may be thawed 5-10X Distribute purified DNA for cloning and expression cloning screens Maintain separate glycerol stocks as source for primary screens (clone identification)

10 Automation of Transformation Implementation 10 Manual Method Addition of 2 ul of DNA to Tubes Containing Competent Cells (10ul) Heat Shock 30 sec at 42 C Ice/Recovery (300 ul) Manual Method used to understand flexibility of each step in traditional transformation protocol DNA concentration range: 10 ng-500 ng/ul DNA volume: 6 ul Elimination of recovery step: maybe Inoculation volumes into 8 well dishes: 5 ul Inoculation to Agar Plate (25 ul)

11 Automation of Implementation Transformation 11 Our Hamilton STAR was configured with a 12-channel variable span pipetter, a 96-well fixed head, a i-swap, heating/cooling modules and a large deck. DNA arrayed into matrix tubes Competent Cells Arrayed in PCR Plate Challenges Cross-contamination Fully-automated process challenging Solutions Optimization of Liquid Class Variables Dispense Height (lower), Speed (Faster), Aspiration Timing (In Place) Pipetting strategy Throughput: 96 samples per hour Transformation efficiency 453/480 Successful 20/27 Successfully upon manual repeat. 7-new DNA required Addition of DNA, heat shock, and inoculation to agar plate occurs on robot Contamination Pre- Optimization Post- Optimization Stephen Monteclaro, Ryan Abraham

12 Automationl Platform Manual Process Automation of Implementation Colony Picking 12 Agar plate containing colonies Toothpicks used to pick a colony Toothpick of with picked colony placed into 96-well culture block with media Platform: RapidPick: Picker with Needle Micro10x: Ability to add media to culture blocks PlateCrane EX: Moves agar plates and culture blocks RSH-4000: Microplate/Culture block sealer. Stephen Monteclaro

13 Automation of Implementation Colony Picking 13 A. Picture out of Focus, no colonies will be identified B. Overpopulated with colonies Software will not identify individual colonies C. Ideal Good separation of colonies, colony size is good & contrast is good D. Ideal Colony identification (artificial colorization) and selection A B C D With out Optimization: 10% growth With Optimization: 99% growth

14 Tina Di Ioia in collaboration with PhyNexus 14 Automation of Mini Preps

15 Mini Automation Purification: Semi-manual Implementation methods 15 Semi manual method - Qiagen 96 well format Typical yields of 5-10 ug Supports gred Ab Engineering 60% Downstream applications sequencing, cloning, transient transfection NAR library management, special projects blocks/week, spikes up to 40+ plates in one day 1 FTE dedicated

16 Automation Considerations Automation How can we automate? Implementation 16 Qiagen: Automating method mechanically possible but not reliable due to high variability in samples PhyTips - Chemistry similar to Qiagen - 200ul PhyTip/1 block per set up (Yvonne Frank s Lab) - Platform had potential for higher throughput mini prep - PhyTips are easy to customize for any liquid handler

17 How can Evaluation we automate? of of PhyTips PhyNexus Is DNA comparable to Qiagen? - Yields, purity, quality Can PhyTip handle typical variation in samples? - Over grown cultures, over centrifuged cultures, fresh pellets Will DNA be suitable for all downstream applications? - Sequencing, transfection, expression cloning assays Automation Implementation 17 Oasis 96 well, 200 ul wide bore sample prep tips, 200 ul PhyTips MEA instrument 12 channel, 1 ml wide bore sample prep tips, 1 ml

18 Automation Implementation Evaluation How can of we PhyTips of automate? PhyNexus : Yield and Criteria Purity 18 Yields need to be 5 10 ug 260/280 Protein contamination, 1.8+ acceptable 260/230 Buffer contamination, 1.8+ acceptable - Significant for mammalian transfection

19 Evaluation of PhyTips : Yield and Purity Automation Implementation Evaluation How can of we PhyNexus automate? Yield & Purity 19 Yields need to be 5 10 ug 260/280 Protein contamination, 1.8+ acceptable 260/230 Buffer contamination, 1.8+ acceptable - Significant for mammalian transfection

20 Automation Evaluation How can we of PhyTips automate? Implementation : Robustness 20

21 Mini How Purification: can Initial we automate? Semi-manual Conclusions methods Automation Implementation 21 Yield, purity, quality, method robustness all look good Next: Downstream applications - Sequencing - Transfection - Expression

22 Mini How Purification: can Initial we automate? Semi-manual Conclusions methods Automation Implementation 22 Yield, purity, quality, method robustness all look good Next: Downstream applications - Sequencing - Transfection - Expression Read Length: >800 Phred Scores: 55

23 Evaluation Forward How can of Transfection we PhyTips automate? : Transfection Efficiency Automation Implementation 23

24 Evaluation of PhyTips : Expression Cloning 24 Lipid Fugene 6 CSP1 Control CSP2 CSP3 Control Bait Protein 1 Bait Protein 2 CSP- Cell Surface Protein Gabriel Quinones

25 Evaluation of PhyTips: Conclusion Automation How can Final we automate? Conclusions Implementation 25 Yields and quality meet our specification 1 ml PhyTip produced a more pure reagent in our experiments than the 200 ul PhyTip (260/230) PhyTips purification is robust. DNA suitable for required downstream applications: sequencing, transfection

26 Final Platform Automation How New can we Platform automate? Implementation - Overview 26 Oasis #1 Oasis #2 Oasis #3 Cell pellets Elution plates Sample prep tips Robot arm Rail 2A 2B 3A 3B Hotel Sample Prep Purification Purification Plate reader In collaboration with dynamic devices

27 Final Platform Automation How New can we Platform automate? Implementation - Overview 27 Control - Method Manager: run each platform - Overload: - coordinates work between platforms - drives rail, hotel, reader 96 position fixed head: O-ring based pipetting head; transitioning to solid mandrel. Buffers pumped into reservoirs, controlled by sensors - Heat wrap around lyses buffer Challenges: - Corrosion - O-ring based head - Imperfect tips holders - Overload

28 To Summarize Automation How can Final we automate? Conclusions Implementation 28 We have implemented a high through put process to manage plasmid based reagents from transformation through purification and storage. We have integrated these systems into informatic infrastructure including registration, inventory management and request.

29 Thanks Automation How New can we Platform automate? Implementation - Overview 29 Genentech Tina Di Ioia Stephen Monteclaro Carmen Chan Lovejit Singh Kurt Schroeder Ryan Abraham Yvonne Franke Gabriel Quinones Qixin Bei Jerry Tang Dan Hascall all of our collaborators PhyNexus Christopher Suh Lee Hoang Dynamic Devices Michael Starace Jeff Horowitz