1. Genomics Technology to Assessing Microbial Activity in the Environment

2. MICRO ARRAY TECHNOLOGY Array technology and how it works How it is likely to be applied and the implications What WILL be gained from its application Advantages and cost benefits

3. Diverse Chemicals and Microorganisms 10 x 10 6 Chemicals –8 x 10 6 Xenobiotic –1 x 10 6 Recalcitrant 0.4 x 10 6 traded at over 50 tonnes per year Toxicological/ biodegradative data on only around 5000- 6000 MICROORGANISMS IN COMPLEX COMMUNITIES IN THE ENVIRONMENT –Difficult to assess their effectiveness –Some cannot be cultured

4. Some soil numbers… Mean particlePer gram Soil typeDiameter (mm)Volume (mm 3 )Number of Particles Surface area (cm 2 ) Very coarse sand2.00-1.004.18 x 10 1 9.0 x 10 1 1.1 x 10 1 Coarse sand1.00-0.505.24 x 10 -1 7.20 x 10 2 2.3 x 10 1 Medium sand0.50-0.256.55 x 10 -2 5.70 x 10 3 4.5 x 10 1 Fine sand0.25-0.108.18 x 10 -3 4.60 x 10 4 9.1 x 10 1 Very fine sand0.10-0.055.24 x 10 -4 7.22 x 10 5 2.27 x 10 2 Silt0.05-0.0026.50 x 10 -5 5.78 x 10 6 4.54 x 10 2 Clay<0.0024.20 x 10 -9 9.03 x 10 10 8.0 x 10 6

5. The ‘tree of life’ rRNA sequence-based phylogenetic tree You are here… Most of the biomass, and most of the diversity is here – there are many missing branches…

6. Assessing microbial diversity These are all nucleic acid (principally rRNA or rDNA) based methods. More direct, analytical methods (ie. FAME analysis / MS fingerprinting) can also be used to estimate diversity. These address only diversity (“Who’s there?”) and not metabolic potential or activity – unless metabolic genes or their transcripts are targeted by gene probes etc. Pure cultures Amplified DNA Nucleic acids Whole Cells FISH / microarray Molecular fingerprinting Gene probe DNA sequencing Sequence database ‘Evenness’ (relative abundance of each taxon) ‘Richness’ (number of unique taxa) Microbial community Clone library

7. Overview of array technology for environmental samples Slide or membrane base Robotic arraymaker makes many multigene arrays. Environmental sample: Extract total DNA or RNA. Use PCR to amplify gene copies (convert RNA to DNA with reverse transcriptase) and apply fluorescent dye label Hybridise to complimentary sequence in array Use laser fluorescence reader to scan slide. Two dyes can be used. One to target genes that identify the bacteria and the other to detect active biodegradation genes. The genes present hybridse to their counterparts on the array and can be detected.

8. Preparation of a DNA Microarray DNA samples may be 1. Oligonucleotides 2. Clones 3. Total DNA 4. PCR products Robotic arm compresses micro samples onto slide Silylated Slides have reactive aldehyde groups and covalently bind tissue, cells or single or double-stranded DNA directly to the glass surface of a high quality microscope slide via the Schiff base aldehyde- amine chemistry (lysine residues of proteins, primary amines of DNA bases, or via synthetic DNA bases bearing amino-modifications).

9. Sample Preparation and hybridisation Complex community of microorganisms Extract total DNA OR RNA Reverse Transcriptase to give DNA PCR to amplify target gene copies Hybridise to complimentary DNA on the array and wash off excess

10. Reading a DNA Microarray after hybridisation Dual labelled fluorescence that can be quantified OR other chromogenic labelling method SLIDE PLACED IN AUTOMATED LASER READER  DATA LOGGED ON COMPUTER

11. INVOLVES APPLICATION OF ADVANCED DNA ARRAY TECHNOLOGY

12. A ‘DNA microarray’ 20,000 features, printed at 170µm pitch. Spot diameter approx 145µm. A different DNA ‘probe’ can be printed on each spot

13. ribosome Bacterial SSU rRNA variability map; red = highly conserved Primary structure (sequence) and secondary structure (folding, loops etc) dictate tertiary (3D) structure of ribosome

14. 16S rDNA gene ~1500bp gene - encodes 16S (small subunit) rRNA molecule Parts of the sequence are conserved among all living things Central metabolism – inherited from the ‘last common ancestor’ 40000+ sequences in the ‘Ribosomal Database Project’ DB Conserved ‘blocks’ can be exploited to ‘amplify’ the gene in vitro Variable regions are used for taxonomy – ‘phylogenetics’ 27f primer 519r primer Highly conserved sequence Variable sequence V1V2V3V5V4V7V6

15. How it is likely to be applied and the implications. Large amount of gene sequence data –Estimated 10 years worth now added in 10 weeks! Gene expression studies –e.g. New drug targets sought Rapid detection of pathogenic bacteria and viruses from complex samples –Organisms detected AND their active genes Rapid detection of biodegrading bacteria in the environment “NO SINGLE AREA OF BIOSCIENCES WITH REMAIN UNAFFECTED BY THIS TECHNOLOGY” (Recent US government report)

16. What will be gained from its application? For the first time it will be possible to analyse a complex microbial population and its relative performance in a SINGLE step. The implications are enormous! This will become the standard diagnostic tool Legislation will follow on from this development THE DESIGN AND SUITABILITY OF THE ARRAYS WILL BE THE MAIN LIMITING STEP IN APPLICATIONS

17. Variability in dioxygenase genes in the Rhodococci (unpublished data)

18. Advantages and cost benefits Revolutionises environmental analysis Analysis of MANY organisms / genes handled in single step. DNA/RNA extracted on site can remain stable High throughput back at the laboratory within one day Fluorescence methodology is sensitive and quantitative We can take advantage of our existing databases and expertise