Difference between revisions of "Part:BBa K374009:Experience"
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The figure shows the change in biomass over time. All three strains have approximately the same growth rates | The figure shows the change in biomass over time. All three strains have approximately the same growth rates | ||
| − | [[Image:Gifsy2_2GFP.png | + | [[Image:Gifsy2_2GFP.png|center|center|thumb|500px|*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter |
| − | *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]] |
| − | *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. |
| − | |||
| − | |||
| − | |||
| − | [[Image: | + | [[Image:Gifsy2_2GFPBiomassvsTime.png|center|center|thumb|500px|*pIGR02_6: pSB4A5 backbone with Gifsy2 pR and pRM promoters. GFP is expressed from the pR promoter |
| − | *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]] |
| − | *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. |
| + | |||
| + | |||
| + | [[Image:Gifsy2_2GFPvsBiomass.png|center|center|thumb|500px|*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==== | ====Conclusion==== | ||
Revision as of 23:36, 27 October 2010
This experience page is provided so that any user may enter their experience using this part.
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how you used this part and how it worked out.
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
The figure shows the change in biomass over time. All three strains have approximately the same growth rates
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.
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.
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
User Reviews
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