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Laboratory : agarose gel & transformation Lecture : reporter genes; transformation

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. Reporter genes monitor

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Laboratory : agarose gel & transformation Lecture : reporter genes; transformation

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  1. Laboratory: agarose gel & transformation Lecture: reporter genes; transformation In-ClassWriting: restriction maps (page 24) HandIn: nothing Read: Appl. Environ. Microbiol. 63: 4920-8, 1997 DueNextClass: report 1 draft

  2. Reporter genes monitor promoter activity or protein expression. Red Fluorescent Protein coding sequence fused to promoter. Visually monitor. Promoter RFP coding sequence

  3. Green Fluorescent Protein fused to GALLS Monitor expression & location Fusion protein Promoter GALLS coding sequence GFP

  4. 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.

  5. Natural Competence: import DNA Due to growth stages; environmental signals. gram-positive: Streptococcus,Bacillus gram-negative: Neisseria, Haemophilus, Vibrio cholera Chemical Competence: CaCl2; RbCl2 Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa)

  6. Chemical transformation: ice-cold CaCl2 or RbCl2 heat shock  plasmid DNA into E. coli. Electroporation: pulses of high voltage  DNA into E. coli & other species.

  7. 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.

  8. Linear DNA usually faster than circular: Circular DNA  2 forms: covalently closed circular (ccc) & open circular (oc). Closed circular DNA  supercoils = twisted telephone cord.

  9. Small supercoiled DNA faster than same length linear. Most supercoiled DNA slower than corresponding linear molecules.

  10. Break in 1 strand of circular DNA  nosupercoiling  "relaxed" or "open" circular DNA  migrates muchmore slowly.

  11. DNA stained with ethidium bromide. Ethidium bromide stacks between bases. Stained DNA + UV  orange light.

  12. 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.

  13. pKN800 A pKN800 B PstI uncut PstI uncut 10.0 8.0 6.0 5.0 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.75

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