Microbial Diversity: Tapping the Untapped

Bioremediation & Microbial Diversity: Applications of Molecular Biological Tools in Studying Novel Physiological Traits Suneel Arjun Chhatre Aug 11, 2009

Microbes: The Earth’s Engine ,[object Object],[object Object],[object Object],[object Object]

Microbial Diversity ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Why is Microbial Diversity Important? ,[object Object],[object Object],[object Object],[object Object]

Role of Carbon ,[object Object],[object Object],[object Object]

The Beginning of Biodegradation ,[object Object],[object Object],[object Object]

[object Object],[object Object]

[object Object],[object Object],[object Object]

Importance of Microbial Diversity ,[object Object],[object Object],[object Object]

[object Object],[object Object],[object Object],[object Object]

Identifying Novel Microbial Catalysis by Enrichment Culture & Screening ,[object Object],[object Object],[object Object],[object Object]

Case Studies: ,[object Object],[object Object],[object Object]

Bioremediation of Oil Spills ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Two Step Treatment Protocol ,[object Object],[object Object]

Goals ,[object Object],[object Object],[object Object],[object Object]

Enrichment of Bacteria ,[object Object],[object Object]

Isolation & Characterization ,[object Object],[object Object],[object Object],[object Object],[object Object]

Designing the Consortium ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Isolate DSS 6 : Colony Characteristics

Isolate DSS 8 : Colony Characteristics

Isolate GSS 3 : Colony Characteristics

Characterization of Isolates on the Basis of Catabolic Pathway Using PCR ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

XylE -Catechol 2-3 Dioxygenase

Efficacy of Consortium for Biodegradation ,[object Object],[object Object],[object Object],[object Object]

Gravimetric Analysis of Various Fractions

Degradation by Individual Members ,[object Object],[object Object],Gas Chromatograph of Crude Oil after 72 hrs

Biosurfactant Production by DSS 6

Imparting Osmotolerance to Consortium ,[object Object],[object Object],[object Object],[object Object],[object Object]

Model Petroleum ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Physical Skimming of Crude Oil ,[object Object],[object Object],[object Object]

Physical Skimming of Crude Oil ,[object Object],[object Object],[object Object],[object Object],[object Object]

Strategies to Study Evolutionary Origin of TCHQ dehalogenase in Sphingobium chlorophenolicum

[object Object],[object Object],PCP Degradation Pathway

[object Object],[object Object],[object Object],The Relationship

[object Object],[object Object],[object Object],[object Object],[object Object]

Insertion of Kanamycin Gene in the Middle of maai Gene pBS + MAI with Kan (pSS-MK) Kan MAI/2 MAI/1 Overlapping PCR MAI+Kan

[object Object],[object Object],[object Object],[object Object],Do We Have the Knockouts?

PCR to verify maai gene with insertion of kanamycin resistance gene in mutants With Long primers With short primers M C6 C8 C9 C10 M C6 C8 C9 C10

Confirmation of knockouts by Phenotype LB+Kan Tyrosine+Minimal Medium

Making a Genomic Library of S. chlorophenolicum ,[object Object],[object Object],[object Object],[object Object]

Optimization and Scale up of Partial Digestion Reaction 4Kb ,[object Object],[object Object],Various enzyme concentration Scale up with right concentration of Sau 3A 4Kb

Vector Preparation and Ligation 4Kb I V ,[object Object],[object Object],[object Object]

[object Object],[object Object],[object Object],4Kb M U C U C U C U C U C U C M U C U C M : Marker U : Undigested C : Cut (digested)

[object Object],[object Object],[object Object],4Kb ~ 650 bp Colony PCR

Experimental Approach II ,[object Object],[object Object],[object Object],[object Object],[object Object]

Primer Design for MAAI ,[object Object],[object Object],[object Object],A B C D A B C D Sequences Blocks Primers

Genomic Library in pSmart ,[object Object],[object Object],PCR Profile of Library Clones

Results : Degenerate PCR ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Site Directed Mutagenesis ,[object Object],[object Object]

Mutations in TCHQ dehalogenase

Quick-change Mutagenesis (Stratagene)

Deletion Mutant ,[object Object],[object Object],[object Object],1 97 108 248

Results PCR for 2 fragments Overlapping PCR and vector pET 21a Product

Colony PCR and Sequencing pcpC Blast 2 with pcpC

Purification of TCHQ dehalogenase ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Substrate Inhibition in TCHQ Dehalogenase ,[object Object],[object Object],[object Object],[object Object]

Substrate Inhibition ,[object Object],[object Object],[object Object],[object Object],[object Object]

Trade Off: Mutant I12A & I12S ,[object Object],[object Object],[object Object]

pH Dependent Protein Expression in Sulfur Oxidizing Bacteria

Deep Sea Thermal Vents ,[object Object],[object Object],[object Object],[object Object]

Cold seeps ,[object Object],[object Object],[object Object],[object Object],[object Object]

Why study them? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Model Organism ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Our reasons to study H. neapolitanus ,[object Object],[object Object],[object Object],[object Object]

“S4”Sulfur Oxidation Pathway

Plan of Action ,[object Object],[object Object],[object Object],[object Object]

Substrate dependent oxygen consumption by Halothiobacillus neapolitanus

Substrate dependent oxygen consumption by Halothiobacillus neapolitanus ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Effect of inhibitors on total O 2 consumption* ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Effect of inhibitors on rate of O 2 consumption ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Substrate:Oxygen Stoichiometry ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

pH Dependent Protein Expression ,[object Object],[object Object]

Summary of Physiology at pH 7 ,[object Object],[object Object],[object Object]

Purification of Proteins involved in Sulfur Oxidation

Cloning & Characterization of Genes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Future Directions ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Acknowledgements ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Acknowledgements ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Future Directions ,[object Object],[object Object]

CODEHOP (Consensus-degenerate Hybrid Oligonucletide Primers) ,[object Object],[object Object],[object Object]

Amplification of maai from Pseudomonas ,[object Object],[object Object],[object Object]

Future Directions ,[object Object],[object Object],[object Object]

Creation of Knockout Mutant by Homologous Recombination Marker pKnock-Km Truncated maai maai from KT 2440 pKnock System

PCRs ,[object Object],maai 1 maai 2 Overlapping PCR ~ 1.6 Kb Kan gene ~ 800 bp

Construction of pSS-MK ,[object Object],[object Object],[object Object],[object Object],[object Object],M V I 3 Kb 1.6 Kb

Expression of Kan Gene in Knockouts ,[object Object],[object Object],3 Kb 1.6 Kb Complete Gene Primers ATG GAG CTG TAC ACC TAT TAC CGT T CC ACC TCG --- --- --- --- --- GCC ATC ATT GGT TGC GAC ATT CAT ATG ATT GAA CAA GAT GGA TTG CAC GCA GGT TCT --- --- --- --- Incomplete Gene Primers (-25 bases) CCA CCT CGT CCT ACC GGG TGC GCA TTG CCC --- --- --- --- --- --- --- CGG CCA TCA TT G GTT GCG ACA TTC AT A TGA TTG AAC A-------- ---

Making Knockouts ,[object Object],[object Object],M C 1 2 3 4 5 6 7 8 9 10 1.6 Kb

Plasmid or Homologous Recombimation? Lane 1-4 : Plasmid DNA prep Lane 5 : uncut pBS vector Lane 6-9 : Genomic DNA prep ,[object Object]

Optimization and Scale up of Partial Digestion Reaction 4Kb ,[object Object],[object Object],Various enzyme concentration Scale up with right concentration of Sau 3A

Vector Preparation and Ligation 4Kb I V ,[object Object],[object Object],[object Object],[object Object]

Site Directed Mutagenesis in TCHQ dehalogenase ,[object Object]

Experimental Approach II ,[object Object],[object Object]

Purification of MAAI from P. putida ,[object Object],Wash 2 Wash 1 Flow through Crude Elute

Purified MAAI on Glutathione Agarose Elute Wash Flow through Load

Sphingobium chlorophinolicum grown on Tyrosine Wash Flow through Supernatent Crude Elutions

Experimental Approach III ,[object Object],Arias-Barran et al , J. Bac 2004

Bioremediation of Contaminated Soil

Microbial Diversity: Tapping the Untapped

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