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Three-Part Small-scale Screening Platform for the Masses PowerPoint Presentation
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Three-Part Small-scale Screening Platform for the Masses

Three-Part Small-scale Screening Platform for the Masses

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Three-Part Small-scale Screening Platform for the Masses

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    2. CESG Protein Production Unified cell-based and cell-free expression pipelines Expression screening in E. coli and wheat germ extract Data captured into Sesame at each step

    3. Modular Vector Design Essential modules bounded by unique restriction sites Promoter, affinity tags, solubility tags, linker combinations In vitro TEV proteolysis of target from other tags Over 70 vector variants

    4. Small-Scale Expression Testing Small-scale expression evaluations (Studier media) SDS-PAGE in slab gels (~2 days processing and analysis) Tested for total expression, fraction soluble and fraction cleaved ~1.5 weeks to evaluate, labor intensive, expensive ($450/ 96 targets) No reliable assessment of target status after TEV proteolysis Predicting large-scale results ~83% accurate (10% false negative and 6% false positive)

    6. Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox

    7. Part 1: A New Modular Vector Design Essential elements bounded by unique restriction sites Promoter, solubility tags, affinity tags, linker combinations Over 70 expression vector variants Linker module altered to contain a TVMV-His8-TEV context Constitutive expression of TVMV protease from vector Altered expression of the lac repressor (LacI) In vivo proteolysis of MBP by TVMV protease (self-cleaving VP62K). Auto-induction medium

    8. First pass Se-Met medium in the production pipeline Factorial design identified favored compositions that were different from original PASM (Studiers media) Expression performance is strongly linked to the level of lac repressor (LacI) expressed from the backbone Part 2: Medium Evolution

    9. Part 3: Simple robotics for the masses Automated Purification System Can the simple Maxwell robotics contribute to our pipeline? Couple the designed vector and the improved expression medium with the new robotics E. coli pipeline: Auto-induction ? in vivo cleavage ? automated Maxwell purification Use it to obtain predictive information of protein behavior in purification, NMR and X-ray structure determination Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox

    10. Automated Analysis System Caliper Lab Chip-90, 1 chip and ~4 hours of screening ($225/96 targets) Accurate determination of the amounts of purified protein targets Preliminary applications Maxwell 16 purified targets MBP-fusions (Se-labeled targets) TEV-treated 15N-labeled targets Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox

    11. LC90 Analysis of Maxwell Screen Various structural genomics targets or controls A mix of the good, the bad and the ugly Target protein detected for all 24 targets In vivo cleavage with GFP as a control worked

    12. New Path to Structure Screening: Micro and Meso-scale protein purification Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox

    13. Meso-Scale Protein Production and Purification for HSQC NMR Analysis Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox Summary: Caliper? Past members thanks Current members, students Supervision, guidance and help from Professor Brian Fox

    14. Acknowledgements Professor Brian G. Fox (section supervisor) Vector Design and FlexiVector Cloning Paul Blommel, Michael Goren, Peter Martin, Kory Seder, Eric Steffan and Russell Wrobel Factorial Evolution of Auto-Induction Media Paul Blommel, Katy Becker and Petar Duvnak CESG Small-scale screening team Lai Bergeman, Mike Cassidy, Ah-Young Lim and Jung (James) Whan Yoon, Jason Bunge (High school student) & Dr Jikui Song (NMR analysis) Past members John Kunert, Megan Riters, Nick Dillion, Rachel Schiesher & Jay Juhjung Chin