Part:BBa_K239009:Experience

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Applications of BBa_K239009

This part was designed to test promoter spy, BBa_K239001. It can be used to detect misfolding of proteins in the periplasm or shear stress in bio-processing.

User Reviews

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The device responded to various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). Possibly it can also be used to detect presence of heterogeneous unfolded proteins in the periplasmic space (e.g. target pharmaceuticals) or to detect high levels of shear stress during cultivation in a bioreactor.

Compare to the related DegP promoter (BBa_K239000), Spy promoter, which is dependent on sigma factor 70, can be induced up to 40 fold by over expression of CpxR. (Bury-Moné et al. 2009) Most importantly, spy promoter showed no respond to stationary phase.

For detailed information please visit [http://2009.igem.org/Team:UCL_London/From_the_lab/Results#Growth_Cruves_Experiments_II UCL_London iGEM Team Wiki Growth_Cruves_Experiments_II]

By iGEM11_UCL_London:

Azo-Sensor

In order to create a BioBrick Azo-sensor, we decided to investigate whether we could repurpose the BBa_K239009 SpyGFP stress sensor submitted by UCL iGEM 2009. We hypothesised that the ‘stress’ caused by the presence of the toxic dyes could activate the GFP promoter and in turn cause result GFP fluorescence with the addition of Azo-dyes.


This assay was performed by growing cells in the presence of the dyes at different concentrations, periodically taking samples and centrifuging to form a cell pellet. The supernatant was discarded and the pellet resuspended in TAE??? buffer. Then, 3 measurements of the GFP fluorescence of the samples were taken and plotted. The results of this assay are below.


Figure 1 - BBa_K239009 SpyGFP stress sensor could be repurposed as a Azo-sensor module in Acid Orange 7 (AO7) dye-contaminated waste waters. Graph showing that E.coli transformed with the BBa_K239009 SpyGFP stress sensor demonstrates GFP fluorescence after incubation in LB media contaminated with AO7 dye. Please note that fluorescence measurements are initially diminished due to the presence of the AO7 dye. However, the increased levels of fluorescence of BBa_K239009 inoculations in low levels of AO7 during the later recordings suggest that GFP expression could be activated through the SpyGFP promoter. Time is shown in hours after incubation. Error bars indicate SEM, n=3.


Although in a very subtle way, low concentrations of Acid Orange 7 seem to have an inducing effect on GFP production of cells transformed with BBa_K239009 SpyGFP sensor around 10 hours after inoculation. This data suggests that this part could potentially be repurposed for detection of dye contaminants in natural or industrial water environments, and further testing with other dyes and concentrations will be carried our.


By UCL 2014