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2 component regulatory systems • Maltose=effector, BUT if signal not DIRECTLY involved, but needs to be transmitted and changed = signal transduction • Sensor protein= – kinase, phosphorylates compounds, – membrane associated • Phosphoryl group transmitted to another regulator IN the cell – Often a DNA binding protein involved in transcription • Many examples, N-fixation, sporulation,chemotaxis Chemotaxis • • • • Attractants decrease rate of autophosphorylation Repellant increased autophosphorylation CheA-CheW=transducer CheY controls switch – cheY-P tumbles, CCW-CW • CheB phosporylated by CheA-P, but slower response than CheY-P • CheB involved methylation – Fully methylated = best for repellants – cheB-P demethylates, occurs when attractants High – Degree of methylation regulates attraction/repulsion Chemotaxis Genetic exchange: transformation Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules – Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) – Some plasmids integrate (F+, Hfr) – Most are double-stranded – About 1- 100kb • Code for: – R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance – Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins – Degradation, tol, nah, • Plasmid copy # • Compatibility (inc) Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules – Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) – Some plasmids integrate (F+, Hfr) – Most are double-stranded – About 1- 100kb • Code for: – R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance – Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins – Degradation, tol, nah, • Plasmid copy # • Compatibility (inc) Avery Experiment Transformation • Competence: ability to be transformed • Steps in transformation – DNA binding and uptake (SS or DS, depending on species) – Integration (recA) • Competence may be induced by electroporation, Ca Mechanism of transformation Transduction • Generalized— low frequency • Specialized— high frequency:requires specific integration Generalized transduction Specialized transduction Specialized transduction Genetic exchange: conjugation Conjugation: early Cell-surface structure Conjugation: middle Conjugation: late Genetic Engineering-basics • Basic steps in cloning – – – – – Restriction-modification enzymes “shot gun” or PCR Ligase Recombination Plasmids (or phage)= vectors Expression vectors • Selection of clones • Looking for a clone with a specific gene – Probes (DNA, RNA or antibody) • Wave of the future: DNA chips or “microarrays”, BAC libraries, automated sequencing etc Genomics • Bioinformatics • Harvesting genes for biotech (Diversa) – Recent Science article • Comparing gene families