Part:BBa_K374008:Experience

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Biolector experiment

The biobrick was cloned into the pSB4A5 plasmid, and a GFP reporter gene was inserted downstream of its pR promoter. The plasmid was transformed into E.coli (DH5 alpha) and measurements were done on this strain using a BioLector microreactor system. The BioLector is a microfermenting system that measures OD and fluorescence simultaneously. From this construct no GFP expression is expected as repression of the pR promoter by the GogR repressor is tight. As a reference the repressor was removed from the construct allowing GFP expression from the pR promoter.

Procedure

• OD600 of an10 mL O/N LB culture with required antibiotics was measured
• Culture was diluted to and OD600 of 0.05
• 1.5 mL of diluted culture was added to each of the 48 wells
• An Adhesive Gas Permeable Seal membrane was applied to the plate
• An Adhesive Seal for Evaporation Reduction on top of the Gas Permeable Seal was applied
• The microplate was incubated in the biolector and fluorescence and light scattering was measured under these conditions:
  • 37 degrees Celcius
  • sampling time 6 times
  • fluorescence gain of 80
  • biomass excitation 620 nm (light scattering)
  • GFP filter was 486nm (ex) / 510nm (em)

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