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

2. Background Biology and Biomedicine are rapidly evolving from 'data-poor' to 'data-rich'-sciences Genomics Transcriptomics ProteomicsMetabolomicsInteractomicsGlycomicsLipidomics

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. www.sys-bio.org/contentimages / 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

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: Molecular structure/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 Roger Kornberg Nobel Prize in Chemistry, 2006 single snapshots; ‘crippled’ complexes containing non- functional substrates; only certain mutants display detectable phenotypes; stability/ viability issues time-scale and/or specificity often difficult to control Low Throughput!

8. 1. Select your Mutagenesis Target …

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

10. 2. Prepare lots of mutants …

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

12. HT Targeted Mutagenesis Sequencing and RNAP Factory X NNN

13. Saturation Mutagenesis - Examples mjA' A822-X mjA' Q823-X T 821 ACC A 822 GCG Q 823 CAG R 820 CGT S 824 AGC G 825 GGT Y 826 TAT V 819 GTG A 818 GCG 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. 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. Archaeal RNAP (Methanocaldococcus jannaschii) Mix in 6M urea and dialyze to assemble native RNAP

16. The RNA Polymerase Factory IN: 1.5 ml bacterial cultures expressing different mutant subunits 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 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. Protein quantitation BCA assay Cell cultures Autoinduction medium Protein extraction FastBreak/Lysonase Chromatography Ion exchange and affinity 96-well Assembly of RNAP 96-well Transcription assays HT electrophoresis E-PAGE48/E-PAGE96 DNA/RNA quantitation Fluorescent assay s Subunit archiving Barcoded storage (-80 o C) Cell density quantitation A 600 assay Viability quantitation Propidium iodide assay Clone archiving Whatman FTA cards RNAP archiving Barcoded storage (-80 o C) Dialysis efficiency Fluorescent assay Specific trx assays Barcoded storage (-80 o C) Non-specific trx assays Fluorescent assay STAGE 2 STAGE 3 STAGE 1

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

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

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

22. 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

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

25. 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

26. Further Details

27. BBSRC

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

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