Part:BBa_K200018:Experience

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Applications of BBa_K200018

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BBa_K200018 1 Not understood Kun

Cells with BBa_K200018 were grown overnight in various different media, and the GFP fluorescence was measured.

After overnight culture, the corrected fluorescence of glucose is almost negligible, showing that glucose represses the PcstA promoter strongly.

For all the other secondary carbon sources, 10% Casamino Acids in M9 shows the highest corrected fluorescence at 22000.

For effective gene expression, not only must the promoter activity be strong, but there must also be enough cells expressing the genes. Therefore, OD600 is also a critical factor.

Due to the exceedingly low OD600 in 0.5% Lactose in M9, the medium cannot be used even though it induces relatively high fluorescence.

For more information, go to the Imperial iGEM 2009 E.ncapsulator project page on [http://2009.igem.org/Team:Imperial_College_London/Wetlab/Results/CRP_and_Media BBa_K200018 testing results]

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[http://2012.igem.org/Team:University_College_London The 2012 iGem UCL team] further characterized the cstA promoter (Part:BBa_K118011) using GFP as a reporter gene (Part:BBa_E0040). E. coli transformed with cstA promoter fused with GFP was grown overnight in LB media, in order to induce the starvation response cells were centrifuged and resuspended in minimal M9 media supplemented with different glucose concentrations (0%, 0.2%, 0.5%, 1.0%, 2.0%, 5%) and chloramphenicol (15 μg/mL). The tested cultures incubated at different concentrations of glucose shown a trend of decreasing GFP expression with raising glucose concentration. Fluorescence was highest at 0% added glucose and lowest at 5%, which implies that the starvation response increases the cstA promoter activity.

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