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

Aug 11, 2009 S. A. Chhatre 2 Microbes: The Earth’s Engine ~4 Billions years Capable of exploiting a vast range of energy sources and thriving in almost every habitat For 2 billion years microbes were the only form of life (all the biochemistries of life evolved) Basic ecosystem processes; biogeochemical cycles and food chains, vital & elegant relationship between themselves and higher organisms

Aug 11, 2009 S. A. Chhatre 3 Microbial Diversity Biodiversity as a source of innovation in biotechnology International Convention on Biological Diversity defines genetic resources as “ genetic material of actual potential value” Microbial Diversity as major resource for biotechnological products and processes Food Biotechnology Metabolites (amino acids, antibiotics, biopharmaceuticals) Enzymes Environmental Biotechnology Biological Fuels

Aug 11, 2009 S. A. Chhatre 4 Why is Microbial Diversity Important? Critical for the sustainability of life on earth, including recycling of elements, maintenance of climate, degradation of wastes Expand the frontiers of knowledge about the limits and strategies of life Largest untapped reservoir of biodiversity Key roles in conservation of higher organisms and in restoration of degraded ecosystems

Aug 11, 2009 S. A. Chhatre 5 Tapping the Untapped?

Aug 11, 2009 S. A. Chhatre 6 Role of Carbon When we study the chemistry of life, carbon is at the center of the action Living things transform carbon-based compounds voraciously, and microbes, as Earth’s most prolific and earliest-evolved life forms, do so most avidly Carbon cycle on Earth is largely dependent on microbiological processes, and biodegradation constitutes one-half of the carbon cycle

Aug 11, 2009 S. A. Chhatre 7 The Beginning of Biodegradation As old as life itself Prebiotic soup of organic molecules that served as the precurosr of the molecules, constituted first life (the ancestral cell) They must also have served as the energy sources (self replication requires energy)

Aug 11, 2009 S. A. Chhatre 8 Explosion of life must have consumed most of the organic molecules in prebiotic soup during the 1st billion years of Earth in the sustainence of first life At that point, the richest source of food for life was other forms of life

Aug 11, 2009 S. A. Chhatre 9 This continues today Microbes produce lipases, proteases, cellulases and ligninases that decmpose living organisms or their remains after death Photosynthesis was an important development on the earth’s surface that allowed much greater biomass production and hence generated more molecules to be biodegraded

Aug 11, 2009 S. A. Chhatre 10 Importance of Microbial Diversity Microbes harbor the greatest biological diversity and play a more important role in maintaining global processes Microbes have been around since the start of the life at least 3.6 billion years ago (macroscopic ~ 1 billion years) Microbes reproduce, and thus evolve new traits faster than macroscopic organisms

Aug 11, 2009 S. A. Chhatre 11 Number of bacteria attached to your body exceeds the entire human population on earth Approximately 5X1030 prokaryotes reside on earth 500,000 species of insects, termites have 1000 sp of bacteria Total number of bacteria in domestic animals is close to 4X1024

Aug 11, 2009 S. A. Chhatre 12 Identifying Novel Microbial Catalysis by Enrichment Culture & Screening One gram of soil contains 109 bacteria, perhaps 10,000 different types Pioneered by Beijerinck & Winogradsky Selective cultivation of one or more bacterial strains obtained from complex mixture such as soil, sludges, water etc. The method relies on using a particular organic compound as the sole carbon source or, less frequently, as the N, S or P source

Aug 11, 2009 S. A. Chhatre 13 Case Studies: Hydrocarbon degrading potential of microbes (Oil Spill Remediation) Reductive Dehalogenation (Degradation of Pesticide, Pentachlorophenol) in Sphingobium cholorophenolicum Sulfur Oxidation Reactions & Acid Tolerance Resposnse in Halothiobacillus neapoitanus

Aug 11, 2009 S. A. Chhatre 14 Bioremediation of Oil Spills EPA (2006): world wide consumption of petroleum was 84,979,000 barrels/day Transportation Oil Spills Disasters Torey Canyon 1967 (38 million gallons) Exxon Valdez 1989 (10 millions gallons plus) Westchester 2000 (567,000 gallons) Hurricane Katrina 2005 (7 millions)

Aug 11, 2009 S. A. Chhatre 15 Two Step Treatment Protocol Containment: Step one is skimming the crude oil from the surface (Sawdust) Mineralization: Step two is biodegradation of crude oil components by using bacterial catabolic properties (Consortium)

Aug 11, 2009 S. A. Chhatre 16 Goals Enrichment, Isolation and Characterization of hydrocarbonoclastic microorganisms Designing a consortium based on their catabolic properties and the composition of crude oil Determination of efficacy of consortium for crude oil/hydrocarbon degradation Osmotolerance (genetic manipulation)

Aug 11, 2009 S. A. Chhatre 17 Enrichment of Bacteria Oil Sludge: Semicontinuous batch reactor fed with crude oil for enrichment of hydrocarbon degraders Serial Dilution & Plating (After six months when COD was 60% lowered)

Aug 11, 2009 S. A. Chhatre 18 Isolation & Characterization Thirty five isolates Three Tier Screening Primary: based on morphology, growth pattern, incubation time Secondary: Antibiotic Sensitivity Tertiary: based on hydrocarbon degradation potential (catechol, dodecane, tetracosane, eicosane, phenanthrene)

Aug 11, 2009 S. A. Chhatre 19 Designing the Consortium Three of the isolates DSS6: Aliphatic degradation, biosurfactant GSS3: Aromatic degrader DSS8: Long chain aliphatic Pseudomonas putida ATCC 102, known for consumption of down stream metabolites Seed culture grown on catechol prior to crude oil degradation

Aug 11, 2009 S. A. Chhatre 20 Isolate DSS6: Colony Characteristics

Aug 11, 2009 S. A. Chhatre 21 Isolate DSS8: Colony Characteristics

Aug 11, 2009 S. A. Chhatre 22 Isolate GSS3: Colony Characteristics

Aug 11, 2009 S. A. Chhatre 23 Characterization of Isolates on the Basis of Catabolic Pathway Using PCR Specific Primers based on the catabolic properties PCR with total DNA of each isolate and control dmpN-Phenol Hydroxylase Pseudomonas sp. (strain CF600) xylE alkB

Aug 11, 2009 S. A. Chhatre 24 alkB-Alkane Hydroxylase From OCT plasmid of Pseudomonas oleovorans

Aug 11, 2009 S. A. Chhatre 25 XylE-Catechol 2-3 Dioxygenase

Aug 11, 2009 S. A. Chhatre 26 Efficacy of Consortium for Biodegradation Gas Chromatography Catechol grown consortium was applied to degrade Crude Oil Control After 72 Hrs.

Aug 11, 2009 S. A. Chhatre 27 Gravimetric Analysis of Various Fractions

Aug 11, 2009 S. A. Chhatre 28 Same methodology but crude oil was subjected to degradation individually Alone, the efficiency was not as high as in group Degradation by Individual Members Gas Chromatograph of Crude Oil after 72 hrs

Aug 11, 2009 S. A. Chhatre 29 Biosurfactant Production by DSS6

Aug 11, 2009 S. A. Chhatre 30 Imparting Osmotolerance to Consortium Pro ‘U’ operon (Dr. Gowrishankar, 1996) Glycine-betaine uptake Subcloned in pMMB206 (a broad host range vector) Growth in presence of 1M Nacl Degradation of Model Petroleum

Aug 11, 2009 S. A. Chhatre 31 Model Petroleum Homogenous mixture of representative hydrocarbons 1-dodecane (C12) 2-naphthalene (Dicyclic) 3-pentadecane (C15) 4-hexadecane (C16) 5-pristane (IS) 6-dibenzothiophene (Hetero) 7-phenanthrene (Tricyclic) 8-eicosane (C20) 9-tetracosane (C24) 10-octacosane (C28 Single peak with Capillary GC)

Aug 11, 2009 S. A. Chhatre 32 Physical Skimming of Crude Oil Alkali Treated sawdust (high temp and pressure) Delignification causes increase in surface area Measured by Methylene Blue Isotherms, Mercury Porosimetry; proved by Scanning Electron Microscopy

Aug 11, 2009 S. A. Chhatre 33 Physical Skimming of Crude Oil Crude Oil was spread over a trough full of water Sprinkling of saw dust Skimming Gravimetric analysis proved ~ 90% removal Cost effective and often necessary

Aug 11, 2009 S. A. Chhatre 34 Strategies to Study Evolutionary Origin of TCHQ dehalogenase in Sphingobium chlorophenolicum

Aug 11, 2009 S. A. Chhatre 35 What is TCHQ Dehalogenase? Reductive dehalogenase, removes 2 chlorine atoms in the PCP degradation pathway in Sphingobium chlorophenolicum PCP Degradation Pathway

Aug 11, 2009 S. A. Chhatre 36 Maleylacetoacetate Isomerase (MAAI):   Catalyzes the isomerization of maleylacetoacetate to fumaryl acetoacetate, a step in the degradation of Phenylalanine and Tyrosine

Aug 11, 2009 S. A. Chhatre 37 The sequence conservation in the active site regions of TCHQ dehalogenase and the known MAA isomerases The ability of TCHQ dehalogenase to isomerize maleylacetone (MA), an analogue of MAA The fact that both are members of zeta class of GST superfamily The Relationship

Aug 11, 2009 S. A. Chhatre 38 Goals Clone, sequence and express the MAA isomerase from S. chlorophenolicum and compare it with TCHQ dehalogenase Determine what type of changes have occurred in order to enhance the dehalogenation reaction In vitro evolution of maai into TCHQ dehalogenase Kinetic studies on dehalogenation

Aug 11, 2009 S. A. Chhatre 39 Experimental Approach I Knocking out maai gene in Pseudomonas putida KT 2440 Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector) Complementing the knockout mutant with genomic library and selecting on tyrosine Preparation of plasmid from the colonies and sequencing

Aug 11, 2009 S. A. Chhatre 40 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

Aug 11, 2009 S. A. Chhatre 41 Genotype : PCR for kanamycin resistant gene inserted in the middle of maai (bigger product) Phenotype : Growth on minimal media+Tyrosine as sole source of carbon Growth on LB+kanamycin plates Do We Have the Knockouts?

Aug 11, 2009 S. A. Chhatre 42 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

Aug 11, 2009 S. A. Chhatre 43 Confirmation of knockouts by Phenotype LB+Kan Tyrosine+Minimal Medium

Aug 11, 2009 S. A. Chhatre 44 Making a Genomic Library of S. chlorophenolicum Optimization of partial digestion of genomic DNA with Sau 3A Scale up the reaction with large quantity of DNA under right conditions Digestion of Vector, dephosphorylation of digested vector Ligation and electroporation

Aug 11, 2009 S. A. Chhatre 45 Optimization and Scale up of Partial Digestion Reaction 4Kb ~ 50 ug of genomic DNA was digested Enzyme concentartion was .05 U/ug of DNA Various enzyme concentration Scale up with right concentration of Sau 3A 4Kb

Aug 11, 2009 S. A. Chhatre 46 4Kb I V Vector Preparation and Ligation Broad host range vector pUCP-Nde (4Kb) BamHI digsetion & depshosphorylation with CIAP Ligation

Aug 11, 2009 S. A. Chhatre 47 Results ~ 28,000 clones Restriction digestion profiles of some of the clones 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)

Aug 11, 2009 S. A. Chhatre 48 Complementation of Knockout with Full Copy of maai Gene Cloning the entire gene (maai) in pUCP-Nde Electroporation of the construct in electrocompetent knockout KT 2440 cells 4Kb ~ 650 bp Colony PCR

Aug 11, 2009 S. A. Chhatre 49 Experimental Approach II Degenerate PCR Amplification of unknown targets related to multiple-aligned protein sequences 2 strategies : Synthesize a pool of degenerate primers containing most or all possible nucleotides Design single consensus primer across the highly conserved region

Aug 11, 2009 S. A. Chhatre 50 Primer Design for MAAI Multiple Sequence Alignment (ClustalW) Block-Maker Codehop A B C D A B C D Sequences Blocks Primers

Aug 11, 2009 S. A. Chhatre 51 Genomic Library in pSmart sau3A digestion of G-DNA Ligation with pre-digested vector PCR Profile of Library Clones

Aug 11, 2009 S. A. Chhatre 52 Results : Degenerate PCR Lane 1-7-14 : Marker Lane 2-8 : Sph. G-DNA Lane 3-9 : Sph. Library Lane 4-10 : KT G-DNA Lane 5-11: PAO1 Lane 6-12: KT Construct Lane 13 : Positive control

Aug 11, 2009 S. A. Chhatre 53 Site Directed Mutagenesis Comparison of TCHQ dehalogenase sequence with known bacterial and eukaryotic MAAIs Mutations in the active site region

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Aug 11, 2009 S. A. Chhatre 56 Mutations in TCHQ dehalogenase

Aug 11, 2009 S. A. Chhatre 57 Quick-change Mutagenesis (Stratagene)

Aug 11, 2009 S. A. Chhatre 58 Sequencing

Aug 11, 2009 S. A. Chhatre 59 Deletion Mutant PCR amplification of the 2 fragments with restriction sites Overlapping PCR for the full fragment Cloning in pET 21a 1 97 108 248

Aug 11, 2009 S. A. Chhatre 60 Results PCR for 2 fragments Overlapping PCR and vector pET 21a Product

Aug 11, 2009 S. A. Chhatre 61 Colony PCR and Sequencing pcpC Blast 2 with pcpC

Aug 11, 2009 S. A. Chhatre 62 Purification of TCHQ dehalogenase Poor Yield (3mg/L) Tedious Prep Three different columns Blue Agarose Mono-Q Superdex

Aug 11, 2009 S. A. Chhatre 63 Substrate Inhibition in TCHQ Dehalogenase Third & Fourth steps Nucleophilic attack of glutathione upon an electrophilic substrate to form a conjugate MAAI & MPI isomerization of double bond, regenenerates glutathione Reductive dehalogenation, 2 equiv of glutathione and results in oxidation to glutathione disulfide

Aug 11, 2009 S. A. Chhatre 64 Substrate Inhibition The substrate primarily binds as TriCHQ- and is rapidly deprotonated to TriCHQ2- at the active site TriCHQ2- is converted to it’s tautomer (TriCHQ* ) which is attacked by glutathione Cys13 then attacks the glutathione conjugate, releasing the reduced product and forming a covalent bond between Cys13 and glutathione Finally, the free enzyme is regenerated by thiol-disulfide exchange reaction with the second molecule of glutathione It is profoundly inhibited by its aromatic substrates

Aug 11, 2009 S. A. Chhatre 65 Trade Off: Mutant I12A & I12S Mutation of Isoleucine 12 to alanine or serine gives and enzyme which is not inhibited by the substrates Weak binding of TriCHQ to ESSG Decrease in rate of dehalogenation

pH Dependent Protein Expression in Sulfur Oxidizing Bacteria

Aug 11, 2009 S. A. Chhatre 67 Deep Sea Thermal Vents Temperatures as high as 404C Depths in 1000’s of meters Pressures >6000 psi Anoxic

Aug 11, 2009 S. A. Chhatre 68 Cold seeps Temperatures 12 to 45 C pH 6.3 to 7.7 Salinity 1200 to 21000 S Eh -380 to -280 mV Depths in 100’s of m

Aug 11, 2009 S. A. Chhatre 69 Why study them? Obvious interest in unique microbial physiologies Species thrive in the presence of high levels of toxic compounds Marine and surface thermal vents long proposed as ‘source of life’ Lateral gene transfer proposed as source for pathogenicity in Proteobacteria Other adaptive responses in Proteobacteria species may also have arisen from horizontal gene transfer Locations range from marine (Mid-Atlantic Ridge) to semi-arid high altitude desert environments (Eddy Co., NM)

Aug 11, 2009 S. A. Chhatre 70 Model Organism Halothiobacillus neapolitanus Isolated from a shallow marine vent Also found in cold seeps and municipal sewers Reduced and partially reduced inorganic sulfur compounds as the sole source of energy Mildly halotolerant, mesophile pH range 8.5 to as low as 3.5

Aug 11, 2009 S. A. Chhatre 71 Our reasons to study H. neapolitanus Wide pH range indicates potential for inducible acid tolerance response (ATR) Chemolithoautotroph Proposed to use the ‘S4’ oxidation pathway Relationship to thermal vent species

Aug 11, 2009 S. A. Chhatre 72 “S4”Sulfur Oxidation Pathway

Aug 11, 2009 S. A. Chhatre 73 Plan of Action Identify sulfur oxidizing activities Establish a baseline for physiology and protein expression Determine pH dependence of physiology and protein expression Establish correlations between physiological changes and expression of individual genes

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Aug 11, 2009 S. A. Chhatre 77 Substrate dependent oxygen consumption by Halothiobacillus neapolitanus

Aug 11, 2009 S. A. Chhatre 78 Substrate dependent oxygen consumption by Halothiobacillus neapolitanus Substrate amount Total O2 consumption O2 consumption rate (nmol) (nmol) (nmolmin-1mg-1) S-2 0 0 0 50 1727 2209 100 33615 2147 S0* 0 0 0 50 8411 558 100 17414 596 S2O3-2 0 0 0 50 997 786 100 1925 825 S4O6-2 0 0 0 50 1556 786 100 3159 825 S306-2 100 0.0 0.0 S5O6-2 100 0.0 0.0 SO3-2 100 0.0 0.0 SO4-2 100 0.0 0.0

Aug 11, 2009 S. A. Chhatre 79 Effect of inhibitors on total O2 consumption* Inhibitor S-2 S0 S2O3-2 S4O6-2 Rotenone 817.8 934.4 100.4  5.7 97.6  6.3 antimycin A 100.511.3 98.1 2.4 85 3.8 100.1 2.7 TTFA 90.14 92  1.9 94.8  3.7 90.3  3.3 myxothiazol 92.24.3 94.7 1.6 99.9 3.8 89.7 6.7 NEM 21.34.5 16.7 4.0 3.9 8.4 23.7 4.9 azide, 0.01mM 1004 1002.2 1005 1003.9 azide, 1mM 84.83.3 84.7 3.1 81.9 6.2 89.6 6.6 cyanide, 0.01mM 1002.2 10017 1009 1006.2 cyanide, 1mM 89.810.4 91.1 7.7 90.0 6.9 92.7 8.9 *values are expressed as percentage of control without inhibitor and without correction for changes in gas solubility due to inhibitors

Aug 11, 2009 S. A. Chhatre 80 Effect of inhibitors on rate of O2 consumption Inhibitor S-2 S0 S2O3-2 S4O6-2 rotenone 54.77% 92 4.6 99.2 2.7 46 8.2 antimycin A 80.85.6% 84 7.7 97.7 3.8 81.2 7.7 TTFA 52.36.1% 55.4 6 100 0.8 88.8  2.7 myxothiazol 79.47.9% 88.4 4.7 98.6 3.0 94.7 4.6 NEM 9.81.4% 1.6 0.9 2.2 1.7 6.1 2.8 azide, 0.01mM 97.3  3.8 94.7 3.8 99.2 1.8 97.8 6.7 azide, 1mM 283.3% 24.4 2.6 27 3.9 25.5 7.0 cyanide, 0.01mM 99.1 2.9 97.2 5.5 98 4.7 97.4 3.0 cyanide, 1mM 30.87.8% 33.2 2.6 31.7 6.8 34 2.3

Aug 11, 2009 S. A. Chhatre 81 Substrate:Oxygen Stoichiometry Substrate mol O2/mol S mol O2/mol e- S-2 ~3.3:1 0.4:1 S0 ~1.5:1 0.6:1 S2O3-2 ~2:1 0.8:1 S4O6-2 ~3:1 0.25:1

Aug 11, 2009 S. A. Chhatre 82 pH Dependent Protein Expression pH 6.5 pH 4.5

Aug 11, 2009 S. A. Chhatre 83 Summary of Physiology at pH 7 Unique electron transport system – terminal oxidase is cyanide insensitive Stoichiometry is not clear Change in expression profile at low pH (ATR)

Aug 11, 2009 S. A. Chhatre 84 Purification of Proteins involved in Sulfur Oxidation

Aug 11, 2009 S. A. Chhatre 85 Cloning & Characterization of Genes N-terminus sequencing of proteins (C-554, C-549, Thiosulfate Oxidase) Primer Designing Genomic Libraries 3-4 Kb 30-40 Kb PCR Cloning & Sequencing Activity Assay, Spectrum

Aug 11, 2009 S. A. Chhatre 86 C-554

Aug 11, 2009 S. A. Chhatre 87 C-554

Aug 11, 2009 S. A. Chhatre 88 Future Directions Quenched oxygen consumption assays Measure NAD(P)/NAD(P)H ratios Measure P/O ratios pH dependence Identification of genes and gene products Real time PCR to verify changes in expression ‘Knock-out’ mutants

Aug 11, 2009 S. A. Chhatre 89 Acknowledgements National Environmental Engineering Research Institute (NEERI)/ Indian Institute of Technology, Roorkee, India Department of Biotechnology (DBT) MCDB/Chemistry, University of Colorado at Boulder, CO NIH, NSF, DOE Chemistry, Eastern New Mexico University, Portales, NM NIH NCRR P20-61480

Aug 11, 2009 S. A. Chhatre 90 Acknowledgements Students Anton Iliuk Ben Goldbaum Eliseo Castillo John Latham Joaquin DeLeon Nalini Anamula Ramu Kakumanu Neela Gamini Dr. Suneel Chhatre Collaborators Sabine Heinhorst Gordon Cannon NIH NCRR P20-61480 ENMU

Aug 11, 2009 S. A. Chhatre 91 Future Directions Purification of MAAI from Sphingobium Characterization of TCHQ dehalogenase mutants

Aug 11, 2009 S. A. Chhatre 92 Acknowledgements Copley Lab Gill lab

Aug 11, 2009 S. A. Chhatre 93 CODEHOP (Consensus-degenerate Hybrid Oligonucletide Primers) Short 3’ degenerate core and a 5’non-degenerate consensus clamp Reducing the length 3’ core decreases the total number of primers Hybridization of the 3’ degenerate core with template is stabilized by non-deg 5’ clamp

Aug 11, 2009 S. A. Chhatre 94 CODEHOP ….

Aug 11, 2009 S. A. Chhatre 95 CODEHOP Output

Aug 11, 2009 S. A. Chhatre 96 Amplification of maai from Pseudomonas Primer design based on only two maai sequences, KT2440 and PAO1 G-DNA as a template Results of combination of Forward 1 primer with 3 different reverse primers

Aug 11, 2009 S. A. Chhatre 97 Quick-change Mutagenesis (Stratagene)

Aug 11, 2009 S. A. Chhatre 98 Future Directions Genomic Library in pBBR1tp Purification of Homogentisate dioxygenase Characterization of TCHQ dehalogenase mutants

Aug 11, 2009 S. A. Chhatre 99 Experimental approach Knocking out maai gene in Pseudomonas putida KT 2440 Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector) Complementing the knockout mutant with genomic library and selecting on tyrosine Preparation of plasmid from the colonies and sequencing

Aug 11, 2009 S. A. Chhatre 100 Creation of Knockout Mutant by Homologous Recombination Marker pKnock-Km Truncated maai maai from KT 2440 pKnock System

Aug 11, 2009 S. A. Chhatre 101 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

Aug 11, 2009 S. A. Chhatre 102 PCRs maai 1 maai 2 ~350 bp Overlapping PCR ~ 1.6 Kb Kan gene ~ 800 bp

Aug 11, 2009 S. A. Chhatre 103 Construction of pSS-MK Gel extraction of right size fragment from PCR Digestion with Hind III and BamH1 Ligation Electropration in XL1Blue cells Plating on LB+Kan and LB+ Amp media M V I 3 Kb 1.6 Kb

Aug 11, 2009 S. A. Chhatre 104 Expression of Kan Gene in Knockouts No growth on LB+Kan plates Several colonies on LB+Amp Plates 3 Kb 1.6 Kb Complete Gene Primers  ATG GAG CTG TAC ACC TAT TAC CGT TCC 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 TTG GTT GCG ACA TTC ATA TGA TTG AAC A-------- ---

Aug 11, 2009 S. A. Chhatre 105 Making Knockouts Electroporation of the constructs and ligation mix into KT 2440 cells 4 colonies showed 1.6 Kb fragment M C 1 2 3 4 5 6 7 8 9 10 1.6 Kb

Aug 11, 2009 S. A. Chhatre 106 Plasmid or Homologous Recombimation? Lane 1-4 : Plasmid DNA prep Lane 5 : uncut pBS vector Lane 6-9 : Genomic DNA prep Looks like we have homologous recombination!

Aug 11, 2009 S. A. Chhatre 107 Making a Genomic Library of S. chlorophenolicum Optimization of partial digestion of genomic DNA with Sau 3A Scale up the reaction with large quantity of DNA under right conditions Digestion of Vector, dephosphorylation of digested vector Ligation and electroporation

Aug 11, 2009 S. A. Chhatre 108 Optimization and Scale up of Partial Digestion Reaction 4Kb ~ 50 ug of genomic DNA was digested Enzyme concentartion was .05 U/ug of DNA Various enzyme concentration Scale up with right concentration of Sau 3A 4Kb

Aug 11, 2009 S. A. Chhatre 109 4Kb I V Vector Preparation and Ligation Broad host range vector pUCP-Nde (4Kb) BamHI digsetion & depshosphorylation with CIAP Ligation

Aug 11, 2009 S. A. Chhatre 110 Results ~ 28,000 clones Restriction digestion profiles of some of the clones 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)

Aug 11, 2009 S. A. Chhatre 111 Complementation of Knockout with Full Copy of maai Gene Cloning the entire gene (maai) in pUCP-Nde Electroporation of the construct in electrocompetent KT 2440 cells 4Kb ~ 650 bp Colony PCR

Aug 11, 2009 S. A. Chhatre 112 Summary Obtain a real knockout (not the contaminant) Ligation reaction Positive control with endogenous promoter

Aug 11, 2009 S. A. Chhatre 113 Experimental Approach I Knocking out maai gene in Pseudomonas putida KT 2440 Making a genomic library of S. chlorophenolicum in a BHRV (Broad host range vector) Complementing the knockout mutant with genomic library and selecting on tyrosine Preparation of plasmid from the colonies and sequencing

Aug 11, 2009 S. A. Chhatre 114 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

Aug 11, 2009 S. A. Chhatre 115 Optimization and Scale up of Partial Digestion Reaction 4Kb ~ 50 ug of genomic DNA was digested Enzyme concentartion was .05 U/ug of DNA Various enzyme concentration Scale up with right concentration of Sau 3A

Aug 11, 2009 S. A. Chhatre 116 4Kb I V Vector Preparation and Ligation Broad host range vector pUCP-Nde (4Kb) BamHI digsetion & depshosphorylation with CIAP Ligation No colonies!

Aug 11, 2009 S. A. Chhatre 117 Complementation of Knockout with Full Copy of maai Gene Cloning the entire gene (maai) in pUCP-Nde, pTZ100, pBBR1tp Electroporation of the construct in electrocompetent knockout KT 2440 cells 4Kb ~ 650 bp Colony PCR

Aug 11, 2009 S. A. Chhatre 118 Site Directed Mutagenesis in TCHQ dehalogenase Comparison of TCHQ dehalogenase sequence with human maleylacetoacetate isomerase

Aug 11, 2009 S. A. Chhatre 119 Mutations in TCHQ dehalogenase

Aug 11, 2009 S. A. Chhatre 120 Experimental Approach II Glutathione agarose (N-linked) does bind TCHQ dehalogenase Does MAAI (P.putida) binds to it?

Aug 11, 2009 S. A. Chhatre 121 Purification of MAAI from P. putida P. putida MAAI with His tag (pET21a) Wash 2 Wash 1 Flow through Crude Elute

Aug 11, 2009 S. A. Chhatre 122 Purified MAAI on Glutathione Agarose Elute Wash Flow through Load

Aug 11, 2009 S. A. Chhatre 123 Sphingobium chlorophinolicum grown on Tyrosine Wash Flow through Supernatent Crude Elutions

Aug 11, 2009 S. A. Chhatre 124 Experimental Approach III Tyrosine degradation cassette in E. coli Arias-Barran et al, J. Bac 2004

Aug 11, 2009 S. A. Chhatre 125 Colony PCR M Mutants C

Aug 11, 2009 S. A. Chhatre 126 Quick-change Mutagenesis (Stratagene)

Aug 11, 2009 S. A. Chhatre 127 Isolate DSS6: Colony Characteristics

Aug 11, 2009 S. A. Chhatre 128 Isolate DSS8: Colony Characteristics

Aug 11, 2009 S. A. Chhatre 129 Isolate GSS3: Colony Characteristics

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Aug 11, 2009 S. A. Chhatre 134 Bioremediation of Contaminated Soil