Application of Molecular Tools in Environmental Engineering

Application of Molecular Tools in Environmental EngineeringCVEN 601February 14, 2017Group 2

NameContributionPresentationAnand PatelSIP MethodSlides 6-15Nidhi RautPCR, RT-PCR & their ApplicationsSlides 16-20Mark RudolphIntroduction/OverviewSlides 2-5

Intro to Microbial EcologyStudy of how organisms on a microscopic scale interact with other organisms and their surroundingsEukaryotesArchaeaBacteria/VirusesWe are interested in determining both the quantity/quality of the microorganisms as well as their actions

Generic TechniquesCulture-DependentEnrichment: Estimate populationPlate Count: Estimate populationCulture-IndependentIsotope Based: Describe activityNucleic Acid Based: Analyzes geneticsFluorescent Staining: Estimate total count or differentiate types

Technique ExamplesPCR (Nucleic Acid)Kinetic Isotope Fractionation (Isotope)DAPI/DTAF/CTC (Fluorescent Staining)T-RFLP (DNA)SIP Method

Key PointsPolymerase chain reaction: make many copies of a specific DNA regionin vitro.Taqpolymerase, and requires DNAprimersAllows 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.

What Is PCR?Goal of PCR : Make enough of the target DNA regionIt last several hours and typically contain 20-30 cycles.This DNA region can be anything the experimenter is interested in.

It is a common laboratory technique used to make many copies of a particular region of DNA.

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Taq PolymeraseDNA polymerase typically used in PCR is calledTaqpolymerase: after the heat-tolerant bacteriumCan tolerate intense heat.High temperature is used repeatedly in PCR todenature DNAPolymerase makes new strands of DNA using existing strands as templates.

(Thermusaquaticus).8

PCR PrimersSequence of nucleotides Provides a starting point for DNA synthesisTaqpolymerase make DNA given aprimer. PCR primers are short pieces of single-stranded DNA, usually around20 nucleotides in length. Two primers are used in each PCR reaction

, 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 copied9

Steps of PCRThe 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.

The key ingredients of a PCR reaction areTaqpolymerase, primers, template DNA(Primers), and nucleotides (DNA building blocks). 10

Steps of PCRThis cycle repeats 25-40 times & takes2-4hoursThe target region can go from just one or a few copies to billions.

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Gel ElectrophoresisSeparates 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 a400base pair (bp) fragment would look like this on a gel

Fragments of DNA are pulled through a gel matrix by an electric current 12

Real Time PCROne 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 increasesFluorescent energy doubles in each cycle and is recorded.https://www.youtube.com/watch?v=kvQWKcMdyS4https://www.youtube.com/watch?v=JmveVAYKylk (1.17)

Quencher rapidly absorbs any light emitted by the Fluorescent molecule.

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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 waterDetection of water-borne microbial pathogens by PCR1.Legionella spp, giardia giardia lamblia, vibrio vulnificus besides L. PneumophilaPurification 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.

Quantification of gene expressionDiagnostic usesMicrobiological usesUses in research in life sciences.Clinical quantification and genotypingEnvironmental remediation applicationsInmicrobiologyandmolecular biology, PCR is used in research laboratories in DNA cloning proceduresDNA sequencing. Diagnosis of microbial infections and epidemiological studies. Important to theagricultural and food industriesas 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 asdehalococcoidesmccartyias well as vinyl chloride reductase functional genes essential to the biodegradation of chlorinated solvents includingtrichloroetheneandtetrachloroethylene.

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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 substrateSeparate biomarkers that contains heavier stable isotopes by ultracentrifugationIsolate them and then analyze

Stable Isotope Probing (SIP)

From National Center of Biotechnology Information

Stable Isotope Probing (SIP)Applications in Environmental Engineering:Biodegradation of harmful compounds in environmentCarbon Cycle StudyRNA-SIP coupled with MAR-FISH has been used to identify acetate utilizing bacterial and archeal population in Anaerobic Digester Sludge

Stable Isotope Probing (SIP)Advantages:Culturally independent methodMost suitable for compounds which are carbon/energy source for microbesCost Effective

Stable Isotope Probing (SIP)Disadvantages:Not suitable for compounds that serve as electron acceptorGives false positives in some casesStakeholder unreliability

ReferencesGoel, 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:10.2175/106143011x13075599869092Cupples, 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-probingMolecular Biological Methods in Environmental Engineering:Goel, Ramesh; Kotay, Shireen M.; Butler, Caitlyn S.; Torres, Csar 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.pdf4Oerther, D.B. & Love, N.G. Re/Views in Environmental Science and Bio/Technology (2003) 2: 1. doi:10.1023/B:RESB.0000022932.74077.5eRanjard, 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.

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