Part:BBa_K374008:Experience
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Applications of BBa_K374008
Biolector experiment
The biobrick was cloned into the pSB4A5 plasmid, and a GFP reporter gene was inserted downstream of its pR promoter (pIGR01_04). 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 (pIGR03_11) 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
The figure shows the change in biomass over time - growth curves for the three different strains. The curves show that the cells without the repressor (pIGR01) grow slower than the cells with the repressor (pIGR03). This could be due to toxicity of GFP.
The figure shows the change in GFP (FU) expression over time (hours). The strain which only contains the Gifsy1 promoters (pIGR01) shows expression of GFP as expected, while the strain containing both the Gifsy1 promoters and the GogR repressor (pIGR03) shows no GFP expression as expected. The DH5-alpha reference shows no expression of GFP which is also expected
The figure shows the normalized GFP expression (FU)/(LSU) over time (hours), or the specific GFP activity over time. When GFP expression is normalized with biomass any variability in GFP expression due to difference in growth is taken into account. We would have expected the activity and thereby the promoter strength, to be constant at all times. However, a constant rate is not reached until after seven hours.
The figure shows GFP (FU) signal according to biomass (SLU). As expected we can see a signal of GFP in the strain without the repressor (pIGR01) and no signal in the other two strains (pIGR03 and DH5-alpha). The linear slopes correspond to the specific activity of GFP expression and thereby also promoter strength. The shift in biomass clearly affects this plot. However, the linear slope before and after the shift are equal.
In the figure above a trend line is added to highlight the linear slope. The linear slope reflects the specific activity of the promoter when the repressor is not present and has been calculated:
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 (pIGR03) compared to the strain without the repressor (pIGR01). The biobrick has also been sequenced and no mutations were seen in neither the promoter regions nor in the repressor. It is also seen that the repression is stable during many generations.
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