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 promoter activity or protein expression.

Promoter RFP coding sequence

Red Fluorescent Protein coding sequence fused to promoter.

Visually monitor.

Green Fluorescent Protein fused to GALLS

Monitor expression & location

Promoter GALLS coding sequence GFP

Fusion protein

Transformation introduces plasmid DNA into E. coli.

You will: 1) transform pKN800 DNA into E. coli, 2) select ampicillin-resistant transformants3) score colonies for luminescence.

Natural Competence: import DNADue 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)

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.

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.

Linear DNA usually faster than circular:

Circular DNA 2 forms: covalently closed circular (ccc) &open circular (oc).

Closed circular DNA supercoils= twisted telephone cord.

Small supercoiled DNA faster than same length linear.

Most supercoiled DNA slower than corresponding linear molecules.

Break in 1 strand of circular DNA no supercoiling "relaxed" or "open" circular DNA migrates much more slowly.

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

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.

pKN800 A pKN800 B

10.08.0

5.0

3.54.0

3.02.5

2.0

1.5

1.0

0.75

6.0

PstI uncut PstI uncut