I. Tools and conceptsa. Analysis of DNAi. Agarose gel electrophoresis can be used to analyze the DNA fragments obtained by treatment with restriction enzymesb. Western bloti. DNA fragments from genomeii. Protein extract subjected to SDS polyacrylamide gel electrophoresisiii. Bands are electrophoretically transferred to a polyvinylidine fluoride (PVDF) membraneA. Membrane is then probed with an antibody directed against the specific proteinB. To visualize the first antibody, the membrane is then probed with a secondary antibody tagged with an enzyme that can break down substrates to emit colorc. Northern bloti. RNA molecules in cellii. RNA extracted from cell and then fractioned by electrophoresisiii. Separated fragments are transferred by simple capillary action onto a membraneA. Membrane hybridized with a small DNA fragment specific for a given mRNA1. Probe is usually radioactively labelediv. RNA’s are then visualized by exposing the membrane to an X-ray film, a process called autoradiographyd. Southern bloti. DNA fragments from genomee. Electrophoretic mobility shift assayi. Allows identification of DNA binding proteinsA. And the conditions when they bindii. Used to determine protein-nucleic acid interactionsA. Mix DNA fragments with proteins, and run on a gelB. Probe with labeled DNA sequence1. Radioactive or fluorescentC. A DNA- protein complex is larger than DNA alone1. Runs a slower on a gelf. Protein purification and analysis methodsi. Separation byA. Size (size exclusion chromatography)B. Charge(ion exchange chromatography)C. Hydrophobicity (hydrophobic interaction chromatography)D. Affinity (immunoaffinity chromatography; metal-binding; tagging)ii. Gene encoding the protein is fused to a DNA sequence encoding a peptide tag that has strong affinity to a small ligand moleculesA. Target ligand is attached to beadsiii. Cell extract containing the tagged protein is passed over the beadsA. Only the tagged protein (fusion protein) will bind to the beads1. Affinity chromatography techniqueiv. There are several commonly used peptide tagsA. One is a series of 6 histidines, called a His6 tag, which tightly binds nickelg. Primer extensions analysisi. Can determine transcript lengthii. Also useful to identify transcriptional start sitesh. DNase protection assaysi. Identifies DNA-protein contact. Expand EMSA analysis and identifies specific nucleotide-protein interactionsi. ChIP-on-chip technologyi. Whole genome DNA-binding analysisii. Determines all the sites in a genome to which a given protein bindsiii. Can identify all of the sequences in a genome to which a given protein will bindiv. First, cells are treated with formaldehyde to cross-link proteins to DNAA. DNA is isolated and sheared into small piecesv. Protein-DNA complexes are “fished” from the proteinA. Chromatin immunoprecipitation of ChIPvi. Cross-links are removed by heat, and the released DBNA fragments are amplified by PCRA. Amplified DNAs are analyzed by microarrayj. PCR methodsi. Identify species in complex microbial communitiesk. Metabolomicsl. MAR-FISHi. MARA. microautoradiographyB. A in situ uptake of specific radiolabeled substrates by individual cells can be observedii. FISHA. Fluorescence in situ hybridizationB. Molecular based technique that allows the in situ phylogenetic identification of cellsiii. MAR-FISHA. Simultaneously examine the phylogenetic identity and the relative or actual specific activity of microorganisms within a complex microbial community at a single-cell levelII. Thermodynamicsa. Science concerned with heat and its relation to energy and workIII. Energy acquisition in bacteria and archaeaa. Printed chartIV. Entropy, enthalpy, and their relation to life and Gibbs Free Energy and biochemical reactionsa. Entropyi. Measure of the randomness of a systemb. Enthalpyi. Measure of the total energy of a thermodynamic systemii. Measure of thermodynamic potentialc. Relation to Gibbs free energyi. DG=DH-TDSii. DG=gibbs free energyiii. DH= change in enthalpy, the heat absorbed or releasediv. TDS= product of temperature and entropy changeV. Energy carriers and electron transfera. Many of the cell’s energy transfer reactions involve energy carriersb. Energy carriersi. Molecules that gain or release small amounts of energy in reversible reactionsii. Examples: NADH and ATPiii. Electron donor is a reducing agentiv. Electron acceptor is an oxidizing agentVI. ATP, NAD(P)H, FADH, etc.a. ATPi. Can transfer energy into cell processes in three different waysA. Hydrolysis releasing phosphate (Pi)B. Hydrolysis releasing pyrophosphate (PPi)C. Phosphorylation of an organic moleculeii. Besides ATP other nucleotides carry energyA. GTP for example1. Provides energy from protein synthesisb. NADHi. Carries 2 or 3 times as much energy as ATP and donates and accepts electronA. NADH is the reduced formB. NAD+ it the oxidized formii. Overall reduction of NAD+ consumes 2 hydrogen atoms to make NADHA. Reaction requires energy input from food moleculesc. FADHi. Coenzyme that can transfer electronsA. FADH2: reduced formB. FAD: oxidized formii. FAD is reduced by 2 electrons and 2 protonsVII. Gibbs free energya. Dg=0, thermodynamic equilibriumb. DG<0, process may go forwardc. DG>0 , the reaction will go in reversed. Determined byi. Molecular stability of reactants and productsii. Entropy changes associated with number and types of productsiii. Concentrations and environmental factorsVIII. How are Gibbs Free Energy and Reduction potential relateda. Reduction potential is the tendency to accept electonsb. Gibbs free energy is the change in heat absorbed or reduced- product of entropy and temp changeIX. What effects delta G and delta Ea. Heat absorbed or releasedb. Temperature and entropy changeX. Energy and electron carriers- what are their characteristicsXI. How do enzymes catalyze metabolic reactions?a. Lower the activation energy allowing rapid conversion of reactants to productsXII. How does ETS enable ATP synthesisa. ETS complexes generate a proton motive force that can drive ATP synthesisXIII. Catabolism: what is meant by the ‘microbial buffet’? What are the three main catabolic pathways?a. Microbial buffet is catabolismb. Main pathwaysi. FermentationA. Partial breakdown of organic food without net electron transfer to an inorganic terminal electron acceptorB. Completion of catabolism without the electron transport system and a terminal acceptorC. Pathways1. HomolacticProduces 2 molecules of lactic acid2.