2 component regulatory systems maltose=effector, but if signal not directly involved, but needs to...
Post on 20-Dec-2015
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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
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)
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
Transduction
• Generalized— low frequency
• Specialized— high frequency:requires specific integration
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