Composite

Part:BBa_K374009:Experience

Designed by: Lisa Blanc Iversen, Maya Friis Kjaergaard, Annemi Jollmann, Anja Sander and Grzegorz Słodkowicz   Group: iGEM10_DTU-Denmark   (2010-10-20)

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Applications of BBa_K374009

Biolector experiment

The biobrick was cloned into the pSB4A5 plasmid, and a GFP reporter gene was inserted downstream of it 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 biomass and fluorescence simultaneously. From this construct no GFP expression is expected as repression of the pR promoter by the GtgR repressor is tight. As a reference the repressor was removed from the construct allowing GFP expression from the pR promoter.

Procedure

  • OD600 of a 10 mL O/N LB culture with required antibiotics was measured
  • Culture was diluted to an 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)


Data

*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter *pIGR04_12: pSB4A5 backbone with BBa K374009 followed by a GFP

The figure shows the change in biomass over time. All three strains have approximately the same growth rates

*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter *pIGR04_12: pSB4A5 backbone with BBa K374009 followed by a GFP

The figure shows the GFP signal over time. As expected we see expression from the strain without the repressor and no signal from the strain with the repressor.


*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter *pIGR04_12: pSB4A5 backbone with BBa K374009 followed by a GFP

The figure shows the normalized GFP expression over time and as expected expression occurs solely in the strains without the repressor. However, we would expect a constant rate at all times, but first after five hours we obtain that.


*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter *pIGR04_12: pSB4A5 backbone with BBa K374009 followed by a GFP‎

The figure shows the GFP signal over biomass. Only the strain without the repressor shows a GFP signal. As for the Gifsy2 we also see a shift in biomass, which can be explained by a metabolic change.

Conclusion

It is clearly shown that the BioBrick is working as expected as there is no expression of GFP in the strain with the repressor (pIGR04).

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