Team:UCL London/From the lab/Results



Copper Experiments

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Note on Copper

Copper is an essential trace metal for all living organisms. Nevertheless, free intracellular copper is very toxic and has a negative impact on most cells even at very low concentrations. Even though the system for the control of copper levels not is fully understood in E.coli two systems, CueR and CusR, have been identified as regulatory systems for copper homeostasis. According to some researchers, increased levels of extra cellular copper may have the effect of denaturing some membrane proteins. (Yamamoto and Ishihama 2005) This has however not yet been proved so there is still a risk that the CpxAR system could be activated by some additional copper mediated mechanism independent of the presence of misfolded proteins in the periplasm.


UCL Cu op 2h.pngFig.1a UCL Cu op night.pngFig.1b CuCl2graph.pngFig.1c' CuSO4graph.pngFig.1d Irongraph.pngFig.1e

Growth Cruves Experiments I

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UCL Growth Curve Expt.pngFig.2a Growth Curve Experiment UCL TetR GC.pngFig.2b Growth Curve of tetR+GFP Device

Experiment I Conclusion

Fig.2a illustrated the growth curves of control (E.coli without any modified plasmids), RPU device (BBa_I20260), degP+GFP device (BBa_K239015), and spy device (BBa_K239009). Fig.2b showed the growth curve of tetR+GFP device (BBa_K239012)

According to the data from Fig.2a, although taking in modified plasmid clearly affected the population of the bacteria, E.coli W3110 reached stationary phase after approximate 8 hours incubation. After about 2 hours of incubation, bacteria turned to start entering exponential phase (log phase). Consequently, some experiments could start at this point to investigate the cell respond at early log phase. Furthermore, the OD of cell cultures reached around 2.0 to 3.0 after 5 hours. This would give a relatively high OD for more clear and strong fluorescent measurements.

Growth Cruves Experiments II

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UCL Fluorescent Bar Chart.png Fig.3a Relative Fluorescent (No stress) with Increasing OD Rpu.pngFig.3b RPU activitity of DegP and Spy promoter

UCL OD&F Control.png UCL OD&F RPU.png UCL OD&F degP.png UCL OD&F spy.png Fig.3c From left to right, the graph illustrated the OD and Relative Fluorescence VS Time of negative control (E.coli without any modified plasmids), RPU device (BBa_I20260), degP+GFP device (BBa_239015), and spy device (BBa_K239009). Please click on the graph to view larger image.

Experiment II Conclusion

Both Fig.3a and Fig.3b indicated that promoter spy is completely silent in all the phases, from lag to stationary phase. More information on BBa_K239001. According to Fig.3a and Fig.3c degP+GFP graph, degP promoter showed weak fluorescence when it reached stationary phase.Note that, at the early lag phase, i.e. 1 hour incubation, degP device showed same level of fluorescence as RPU, which also suggested that degP promoter was fully activated in last stationary phase and it was adapting new environment in the lag phase of this incubation. Additionally, from other testing experiences of our project, the overnight deP+GFP cell cultures gave strong green fluorescence. The possible reasons are clearly explained under the BBa_L239000 parts page.

As a consequence, promoter degP works most efficient and accurate at early and mid exponential phase. Whereas, promoter spy is suitable to detect stress under any other conditions, especially in stationary phase.

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