Automation of Complex Procedures in Molecular Biology

Robert Weinzierl Imperial College London

BackgroundBackground• Biology and Biomedicine are rapidly evolving

from 'data-poor' to 'data-rich'-sciences

Genomics

Transcriptomics

ProteomicsMetabolomics

Interactomics

Glycomics

Lipidomics

BackgroundBackground

• 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

www.sys-bio.org/contentimages/

BioinformaticsBioinformatics

• 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

New ChallengesNew 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!

Molecular Molecular Structure/Function Structure/Function

RelationshipsRelationships

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.

Structure/Function Studies 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 KornbergNobel 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 controlLo

w Thr

ough

put!

1. Select your Mutagenesis Target …

PDB #1R5U and 1I6H

The Bridge HelixThe Bridge Helix

2. Prepare lots of mutants …

Mutagenesis with

one oligonucleotide

Amplification and transfer into expression host

High-throughput plasmid purification & sequencing

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HT Targeted MutagenesisHT Targeted Mutagenesis

Sequencing and

RNAP Factory

XNNNNNN

Saturation Mutagenesis - ExamplesSaturation Mutagenesis - Examples

mjA' A822-X

mjA' Q823-X

T821

ACC

A822

GCG

Q823

CAG

R820

CGT

S824

AGC

G825

GGT

Y826

TAT

V819

GTG

A818

GCG

• The likelihood of obtaining particular substitutions depends on the frequency of codons within the genetic code

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

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

The RNA Polymerase FactoryThe 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.

Protein quantitationBCA assay

Cell culturesAutoinduction medium

Protein extractionFastBreak/Lysonase

ChromatographyIon exchange and affinity

96-well Assembly of RNAP

96-well Transcription assays

HT electrophoresisE-PAGE48/E-PAGE96

DNA/RNA quantitation

Fluorescent assaysSubunit archiving

Barcoded storage (-80oC)

Cell density quantitationA600 assay

Viability quantitationPropidium iodide assay

Clone archivingWhatman FTA cards

RNAP archivingBarcoded storage (-80oC)

Dialysis efficiencyFluorescent assay

Specific trx assaysBarcoded storage (-80oC)

Non-specific trx assaysFluorescent assay

STAGE

2

STAGE

3

STAGE

1

E-PAGE 96

Bacterial Cell Density

Subunit Concentration

% Non-viable Cells

Expression Strain Identity

Individual

2D Barcode

Parallel Robotic Assembly of 96 Parallel Robotic Assembly of 96 Different RNAPsDifferent RNAPs

DialysisMembrane

Urea-freeBuffer

Waste

Magnetic Stirrer

Hours

[Urea](Molar)

wt

mjRNAP (wildtype)

V819A

mjRNAP A'-V819A

R820AmjRNAP A'-R820A

etc. (x96!)

T821A

mjRNAP A'-T821A

Fu

nction

al Assays

• 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

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

ConclusionsConclusions

• 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

Further DetailsFurther Details

BBSRC

mjRNAP

neRNAP

Fluoride salts

wt activity

High-throughput Transcription AssaysHigh-throughput Transcription Assays

neRNAP is a neRNAP is a ''Fluorophile Fluorophile ''

Tris-Ac Tris-HCl Tris-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