Transcript 2 component regulatory systems

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
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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
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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