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Laboratory: agarose gel & transformation Lecture: reporter genes; transformation In-Class Writing: restriction maps (page 24) Hand In: nothing Read: Appl. Environ. Microbiol. 63: 4920-8, 1997 Due Next Class: report 1 draft
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Reporter genes monitor promoter activity or protein expression. Promoter RFP coding sequence Red Fluorescent Protein coding sequence fused to promoter. Visually monitor.
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Green Fluorescent Protein fused to GALLS Monitor expression & location Promoter GALLS coding sequence GFP Fusion protein
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Transformation introduces plasmid DNA into E. coli. You will: 1) transform pKN800 DNA into E. coli, 2) select ampicillin-resistant transformants 3) score colonies for luminescence.
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Natural Competence: import DNA Due to growth stages; environmental signals. gram-positive: Streptococcus, Bacillus gram-negative: Neisseria, Haemophilus, Vibrio cholera Chemical Competence: CaCl 2 ; RbCl 2 Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa)
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Chemical transformation: ice-cold CaCl 2 or RbCl 2 heat shock plasmid DNA into E. coli. Electroporation: pulses of high voltage DNA into E. coli & other species.
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Agarose gel electrophoresis separates DNA by size & structure. DNA negative charge migrates from cathode (negative, black lead) to anode (positive, red lead). Agarose = molecular sieve Retards long DNA more than short DNA.
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Linear DNA usually faster than circular: Circular DNA 2 forms: covalently closed circular (ccc) & open circular (oc). Closed circular DNA supercoils = twisted telephone cord.
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Small supercoiled DNA faster than same length linear. Most supercoiled DNA slower than corresponding linear molecules.
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Break in 1 strand of circular DNA no supercoiling "relaxed" or "open" circular DNA migrates much more slowly.
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DNA stained with ethidium bromide. Ethidium bromide stacks between bases. Stained DNA + UV orange light.
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Estimate size of restriction fragments. Compare mobility to size standards. Make standard curve: log [size] = Y-axis migration distance = X-axis for standard bands. Measure migration of restriction fragments; interpolate from standard curve to estimate sizes.
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pKN800 ApKN800 B 10.0 8.0 5.0 3.5 4.0 3.0 2.5 2.0 1.5 1.0 0.75 6.0 PstI uncut
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