Application of Molecular Biotechnologies to Remediation

1. Application of Molecular Biotechnologies to Remediation Shu-Chi Chang, Ph.D., P.E., P.A. Assistant Professor1 and Division Chief2 1Department of Environmental Engineering 2Division of Occupational Safety and Health, Center for Environmental Protection and Occupational Safety and Health National Chung Hsing University Wednesday, June 13, 2007

2. Categories • Molecular biological methods • Biochemical methods • Microbiological methods

3. Molecular biological methods • PCR based • A PCR animation from “Molecular Biology of the Cell” • Probe hybridization

4. PCR based • ARDRA (amplified ribosomal DNA restriction analysis): Separates amplified 16S molecules by restriction patterns • DGGE (denaturing gradient gel electrophoresis): Separates amplified 16S molecules by %G-C content • TGGE (temperature gradient gel electrophoresis): Separates amplified 16S molecules by %G-C content; • T-RFLP (terminal-restriction fragment length polymorphism): Separates amplified 16S molecules by restriction patterns • LH-PCR (length heterogeneity polymerase chain reaction): Separates amplified 16S molecules by length • RISA (ribosomal intergenic spacer analysis): Separates amplified 16S-23S intergenic region by length • SSCP (single-strand conformation polymorphism): Separates amplified 16S ssDNA by sequence-dependent higher order structure • RAPD (randomly amplified polymorphic DNA): Sequence-independent profiling based on random PCR priming, • Sequencing of cultured isolates: Sequencing of PCR amplicons derived from cultured isolates • Functional PCR: Several PCR-based analyses using amplified catabolic genes; indirect functional assay • Direct cloning and sequencing: Direct sequencing of isolated and cloned fragments

5. ARDRA • Amplify community rDNA • Add combinations of restriction enzymes • Assumption: if right enzymes were used, each species will have a unique pattern (fingerprint). However, it is hard to differentiate from each other. Usually only one fingerprint for one community • BY incorporating probe hybridization, more detail information can be obtained • Disadvantage: need optimized combination of restriction enzymes. • Advantage: fast and cost-effective

6. DGGE • Different G-C contents render different mobility in DNA-denaturing gel which is prepared to have a concentration gradient of denaturant. • Probably most widely applied method for community characterization. • Limitation • Need to optimize the gradient and electrophoresis duration • DNA fragment < 500bp • Need large quantity of DNA • Statistical method may help to resolve some problems associated with DGGE.

7. T-RFLP • Modified form of ARDRA using fluorescent PCR primers • Limitation of database (only prokaryotic) • Can only observe 50 or so populations • Sensitivity ~0.5% • Potential bias from PCR • Probably more quantitative than other methods

8. RISA • Ribosomal intergenic region • Utilizing natual variability of rrl operon in rRNA • Can be used to distinguish different strains and closely related species • Rapid and simple but biases from PCR and secondary structure.

9. RAPD • Is able to generate a unique set of amplicons for each species. • random short PCR primer • Usually 5~15 sets per species • Cannot be complemented by other method

10. Comparison of methods

11. Probe hybridization • General probe hybridization: Identifies presence of desired sequences using labeled probes • DNA microarrays: Extremely high-throughput multiple probe hybridization

12. Probe hybridization • Purposes • Presence of various taxanomic groups • Measure relative abundance • Determine their spatial distribution • Type • FISH • CISH • CARD-FISH • MAR-FISH

13. Probe hybridization • Advantages • Great flexibility • Rapid and low cost • Good specificity, usually • Can aim at multiple targets • Disadvantages • Probe design ->mismatch • Sensitivity

14. DNA microarray

15. Microarray data analysis

16. Microarray • Related areas • Bioinformatics : Online Services : Gene Expression and Regulation at the Open Directory Project • Gene Expression : Databases at the Open Directory Project • Gene Expression : Software at the Open Directory Project • Data Mining : Tool Vendors at the Open Directory Project • Notable companies • Affymetrix • Agilent Technologies • CombiMatrix • Eppendorf • Nanogen

17. Biochemical methods • DNA composition and kinetics assays • DNA reassociation kinetics: Estimates sample diversity based on rate of reassociation of denatured DNA • Bisbenzimidazole-CsCl-gradient fractionation: DNA fractionnation based on %GC content • Community DNA hybridization: Estimates relative similarity of two communities by cross hybridization kinetics • Metabolic assays • Metabolomics: Emerging technique to profile total metabolites produced by a community • Lipid analyses • Quinone profiling: Culture-independent community profile based on distribution of quinones • PLFA (phospholipids fatty acids) + FAME (fatty acid methyl esters): Culture-independent community profile based on distribution of various membrane lipids

18. Metabolomics • Systematic study of the unique chemical fingerprints that specific cellular processes leave behind • mRNA gene expression data and proteomic analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell.

19. PLFA

20. PLFA

21. PLFA

22. Microbiological methods • Metabolic assay • CLPP (community-level physiological profiling): Creates a profile of substrates metabolized by the microbial community • Cell counting techniques • Direct cell counting: Microscopic counting of stained cells • Indirect cell counting: Counting of a culturable subset of the microbial community • Morphological counting: Microscopic identification and enumeration of the morphotypes in an environmental sample • Flow cytometry and cell sorting: Physically separates microbial assemblages on the basis of measurable properties,

23. CLPP • BiologTM

24. Side Scatter : Blue Fluorescence 1: Green Fluorescence 2: Yellow Side scatter light Fluorescence 3: Red Fluorescence 4: Dark red Light source Forward scatter light Forward scatter : Blue Basics of flow cytometry

25. 635nm Red Laser Basics of flow cytometry • Three major modules: Optics, Electronics, and Microfluidics. Fluorescence 1 Side Scatter 530/30 Fluorescence 2 488/10 Fluorescence 4 585/42 661/16 Fluorescence 3 670LP 488nm Blue Laser FSC 488/10

26. Side scatter light SSC FL1 FL2 R2 FL4 FL3 Light source Forward scatter light R1 FSC Flow cytometry output High FL1: Green Fluorescence Low Side scatter light Opaque Transparent