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Automation of Complex Procedures in Molecular Biology Robert Weinzierl Imperial College London

Automation of Complex Procedures in Molecular Biology Robert Weinzierl Imperial College London. Background. Biology and Biomedicine are rapidly evolving from 'data-poor' to 'data-rich'-sciences. Genomics. Genomics. Transcriptomics. Proteomics. Interactomics. Glycomics. Lipidomics.

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Automation of Complex Procedures in Molecular Biology Robert Weinzierl Imperial College London

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  1. Automation of Complex Procedures in Molecular BiologyRobert Weinzierl Imperial College London

  2. Background • Biology and Biomedicine are rapidly evolving from 'data-poor' to 'data-rich'-sciences Genomics Genomics Transcriptomics Proteomics Interactomics Glycomics Lipidomics Metabolomics

  3. Background • The new technologies share a common property: • High-throughput technologies are essential for describing and cataloguing the complexities of biological systems • Basic Science: important for functional insights • Diagnostics: allows detection of normal and abnormal states High-Throughput

  4. Bioinformatics • The large data sets reveal functional correlations between individual systems elements • Enhanced understanding of complex systems • Computer simulations can be used to test predictions against real data sets www.sys-bio.org/contentimages/

  5. New Challenges • ‘Systems approaches’ have been successfully applied to a number of biological systems • There are, however, major gaps at the most fundamental level: Molecularstructure/function relationships are still poorly understood!

  6. Molecular Structure/Function Relationships Catalytic Center of RNA Polymerase II Tan, L., Wiesler, S., Trzaska, D., Carney, H.C. and Weinzierl, R.O.J. (2008). Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J. Biol. 7, 40.

  7. Structure/Function Studies • Structural Approaches: • X-ray crystallographic analysis of bacterial, archaeal and yeast RNAPs • Genetic Approaches: • Isolation of random mutants, especially in bacterial and yeast RNAPs, displaying detectable phenotypes • Biochemical Approaches: • Chemical cross-linking studies; nucleotide analogs • single snapshots; ‘crippled’ complexes containing non-functional substrates; Low Throughput! Roger Kornberg Nobel Prize in Chemistry, 2006 • only certain mutants display detectable phenotypes; stability/ viability issues • time-scale and/or specificity often difficult to control

  8. 1. Select your Mutagenesis Target …

  9. The Bridge Helix PDB #1R5U and 1I6H

  10. 2. Prepare lots of mutants …

  11. Mutagenesis with one oligonucleotide Amplification and transfer into expression host http://www.suta-raito.com/Neopets.shtml High-throughput plasmid purification & sequencing

  12. X NNN NNN HT Targeted Mutagenesis Sequencing and RNAP Factory

  13. Saturation Mutagenesis - Examples A 818 GCG V 819 GTG R 820 CGT T 821 ACC A 822 GCG Q 823 CAG S 824 AGC G 825 GGT Y 826 TAT mjA' A822-X mjA' Q823-X • The likelihood of obtaining particular substitutions depends on the frequency of codons within the genetic code

  14. 3. Prepare mutant subunits and incorporate them into an intact enzyme …

  15. Mix in 6M urea and dialyze to assemble native RNAP Archaeal RNAP (Methanocaldococcus jannaschii) Werner, F., and Weinzierl, R.O.J. (2002). A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol. Cell 10, 635-646. Ouhammouch, M., et al. (2004).A fully recombinant system for activator-dependent archaeal transcription. J. Biol. Chem. 279, 51719-51721. Werner, F., and Weinzierl, R.O.J. (2005). Direct modulation of RNA polymerase core functions by basal transcription factors. Mol. Cell. Biol. 25, 8344-8355.

  16. OUT: Purified and characterized mutant recombinant subunits OUT: Recombinant RNAPs assembled with the mutant subunits purified from bacterial cultures OUT: Expression plasmids archived for long-term storage IN: 1.5 ml bacterial cultures expressing different mutant subunits The RNA Polymerase Factory OUT: High-throughput activity measurements from in vitro transcription results Nottebaum, S., Tan, L., Trzaska, D., Carney, H.C., and Weinzierl, R.O.J. (2008). The RNA polymerase factory: a robotic in vitro assembly platform for high-throughput production of recombinant protein complexes. Nucl. Acids Res. 36, 245-252.

  17. Cell density quantitation A600 assay Cell cultures Autoinduction medium Clone archiving Whatman FTA cards Viability quantitation Propidium iodide assay STAGE 1 Protein extraction FastBreak/Lysonase Protein quantitation BCA assay Chromatography Ion exchange and affinity Subunit archiving Barcoded storage (-80oC) DNA/RNA quantitation Fluorescent assays HT electrophoresis E-PAGE48/E-PAGE96 STAGE 2 96-well Assembly of RNAP Dialysis efficiency Fluorescent assay RNAP archiving Barcoded storage (-80oC) STAGE 3 96-well Transcription assays Non-specific trx assays Fluorescent assay Specific trx assays Barcoded storage (-80oC)

  18. Bacterial Cell Density % Non-viable Cells Subunit Concentration Expression Strain Identity E-PAGE 96 Individual 2D Barcode

  19. Parallel Robotic Assembly of 96 Different RNAPs [Urea] (Molar) Hours Dialysis Membrane Urea-free Buffer Waste MagneticStirrer

  20. wt V819A R820A T821A mjRNAP (wildtype) mjRNAP A'-V819A Functional Assays mjRNAP A'-R820A mjRNAP A'-T821A etc. (x96!)

  21. Complete mutagenesis data for 17 successive amino acid positions reveals a wide variety of phenotypes • Identification of the most informative mutants for revealing reaction mechanism • Approach also identifies mutants that do not have a significant effect

  22. Q823 Q823 Side Chain Requirements • (quite variable) • direct control of catalytic rate A822 A822 Side Chain Requirements • (variable; large hydrophilic side chains [Q, R] acceptable) T821

  23. Conclusions • Robotic applications in Molecular Biology do not make life easier – they expand what can be done • Complex experiments, once automated, can produce more results than humanly (psychologically!) possible • Repeats and multiple samples provide statistical measure of accuracy/reproducibility • The collection of large systematic data sets allow the unbiased detection of unexpected phenomena

  24. Further Details

  25. BBSRC

  26. High-throughput Transcription Assays mjRNAP neRNAP Fluoride salts wt activity

  27. neRNAP is a 'Fluorophile ' wt activity [10x] Potassium fluoride + 0.1M Tris-HCl pH8.5 Ammonium fluoride + 0.1M Tris-HCl pH8.5 Potassium fluoride +25% PEG3350 Ammonium fluoride +25% PEG3350 Tris-Ac Tris-HCl Tris-Ac +PEG Tris-HCl +PEG

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