Application of molecular tools in environmental engineering (with references)
- 1. Application of Molecular Tools in Environmental Engineering CVEN 601 February 14, 2017 Group 2
- 2. Name Contribution Presentation Anand Patel ● SIP Method Slides 6-15 Nidhi Raut ● PCR, RT-PCR & their Applications Slides 16-20 Mark Rudolph ● Introduction/Overview Slides 2-5
- 3. Intro to Microbial Ecology • Study of how organisms on a microscopic scale interact with other organisms and their surroundings • Eukaryotes • Archaea • Bacteria/Viruses • We are interested in determining both the quantity/quality of the microorganisms as well as their actions
- 4. Generic Techniques Culture-Dependent • Enrichment: Estimate population • Plate Count: Estimate population Culture-Independent • Isotope Based: Describe activity • Nucleic Acid Based: Analyzes genetics • Fluorescent Staining: Estimate total count or differentiate types
- 5. Technique Examples • PCR (Nucleic Acid) • Kinetic Isotope Fractionation (Isotope) • DAPI/DTAF/CTC (Fluorescent Staining) • T-RFLP (DNA) • SIP Method
- 6. Key Points Polymerase chain reaction: make many copies of a specific DNA region in vitro. Taq polymerase, and requires DNA primers Allows many copies of the target region to be produced. PCR has many research and practical applications. Routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.
- 7. What Is PCR? • Goal of PCR : Make enough of the target DNA region • It last several hours and typically contain 20-30 cycles. • This DNA region can be anything the experimenter is interested in.
- 8. Taq Polymerase • DNA polymerase typically used in PCR is called Taq polymerase: after the heat-tolerant bacterium • Can tolerate intense heat. • High temperature is used repeatedly in PCR to denature DNA • Polymerase makes new strands of DNA using existing strands as templates.
- 9. PCR Primers • Sequence of nucleotides • Provides a starting point for DNA synthesis • Taq polymerase make DNA given a primer. • PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length. • Two primers are used in each PCR reaction
- 10. Steps of PCR The ingredients are assembled in a tube and put through repeated cycles. • Denaturation: To separate, or denature, the DNA strands. Provides single-stranded template for the next step. • Annealing : Cool the reaction so the primers can bind to their complementary sequences. • Extension: Raise the reaction temperature: polymerase synthesizing new strands of DNA.
- 11. Steps of PCR • This cycle repeats 25-40 times & takes 2-4 hours • The target region can go from just one or a few copies to billions.
- 12. Gel Electrophoresis • Separates DNA fragments according to size. • Agarose gel and stain it with a dye which makes it visible. • Brighter band = more copies of your target created. • PCR reaction producing a 400 base pair (bp) fragment would look like this on a gel
- 13. Real Time PCR • One end of nucleotides probe is labelled covalently with a fluorescent molecule and another with a quencher molecule. • Process of making new DNA: Disassemble the double stranded path( F-Q Probe). • Marker get separated; release Fluorescent energy increases • Fluorescent energy doubles in each cycle and is recorded. https://www.youtube.com/watch?v=kvQWKcMdyS4 https://www.youtube.com/watch?v=JmveVAYKylk (1.17)
- 15. Environmental Remediation Applications Detection of indigenous microorganisms 1. Detection of degrading microorganisms • Bioremediation • Biodegradation 2. Identification of Microorganisms in biofilms Detection of indicator microorganisms in water Detection of water-borne microbial pathogens by PCR 1.Legionella spp, giardia giardia lamblia, vibrio vulnificus besides L. Pneumophila • Purification of nucliec acid from soil and sediments, and micro-organisms in water. • Quantify functional genes involved in the biodegradation of various hydrocarbons. • Quantify gene expression and identify biodegradation activity.
- 16. Stable Isotope Probing (SIP) Procedure: • Prepare a substrate by adding heavier stable isotopes (13C (Most Popular), 15N, 18O) • Extract biomarkers (DNA/RNA, Proteins, Phospholipid Fatty Acid) from the substrate • Separate biomarkers that contains heavier stable isotopes by ultracentrifugation • Isolate them and then analyze
- 17. Stable Isotope Probing (SIP) From National Center of Biotechnology Information
- 18. Stable Isotope Probing (SIP) Applications in Environmental Engineering: • Biodegradation of harmful compounds in environment • Carbon Cycle Study • RNA-SIP coupled with MAR-FISH has been used to identify acetate utilizing bacterial and archeal population in Anaerobic Digester Sludge
- 19. Stable Isotope Probing (SIP) Advantages: • Culturally independent method • Most suitable for compounds which are carbon/energy source for microbes • Cost Effective
- 20. Stable Isotope Probing (SIP) Disadvantages: • Not suitable for compounds that serve as electron acceptor • Gives false positives in some cases • Stakeholder unreliability
- 21. References Goel, R., Kotay, S. M., Butler, C. S., Torres, C. I., & Mahendra, S. (2011). Molecular Biological Methods in Environmental Engineering. Water Environment Research,83(10), 927-955. doi:10.2175/106143011x13075599869092 Cupples, A. M. (2016). Contaminant-Degrading Microorganisms Identified Using Stable Isotope Probing. Chemical Engineering & Technology,39(9), 1593-1603. doi:10.1002/ceat.201500479 (n.d.). Retrieved February 17, 2017, from https://www.coursera.org/learn/natural-attenuation-of-groundwater- contaminants/lecture/83aUM/stable-isotope-probing Molecular Biological Methods in Environmental Engineering:Goel, Ramesh; Kotay, Shireen M.; Butler, Caitlyn S.; Torres, César I.; Mahendra, Shaily,source: Water Environment Research, 2011 Literature Review, pp. 927-955(29) http://home.eng.iastate.edu/~tge/ce421-521/shuhao.pdf:http://home.eng.iastate.edu/~tge/ce421-521/shuhao.pdf4 Oerther, D.B. & Love, N.G. Re/Views in Environmental Science and Bio/Technology (2003) 2: 1. doi:10.1023/B:RESB.0000022932.74077.5e Ranjard, Lionel, Franck Poly, and Sylvie Nazaret. "Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment." Research in Microbiology 151.3 (2000): 167-177.
- 22. Questions?
Editor's Notes
- #8: It is a common laboratory technique used to make many copies of a particular region of DNA.
- #9: (Thermus aquaticus).
- #10: , and they are designed so that they flank the target region . That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied
- #11: The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA(Primers), and nucleotides (DNA building blocks).
- #13: Fragments of DNA are pulled through a gel matrix by an electric current
- #14: Quencher rapidly absorbs any light emitted by the Fluorescent molecule.
- #16: Quantification of gene expression Diagnostic uses Microbiological uses Uses in research in life sciences. Clinical quantification and genotyping Environmental remediation applications In microbiology and molecular biology, PCR is used in research laboratories in DNA cloning procedures DNA sequencing. Diagnosis of microbial infections and epidemiological studies. Important to the agricultural and food industries as a valuable alternative to traditional detection methods. Forensics laboratories. 1.Legionella spp. Is A water-borne microbial pathogen and can cause legionnaires' disease in humans via aerosol. 1.Legionella spp. Is A water-borne microbial pathogen and can cause legionnaires' disease in humans via aerosol. 2. PCR detection of giardia another microbial pathogen, giardia lamblia, causes defined waterborne diarrhea in the united states and in many other parts of the world. 3. PCR detection of vibrio vulnificus besides L. Pneumophila, another important marine water-borne microbial pathogen, vibrio vulnificus, which can cause fatal infections in humans Qpcr is used to detect and quantify bacteria such as dehalococcoides mccartyi as well as vinyl chloride reductase functional genes essential to the biodegradation of chlorinated solvents including trichloroethene and tetrachloroethylene.