Difference between revisions of "Part:BBa K2097000"
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| + | Top10 E. coli are grown in unaltered M9 minimal media with chloremphenicol (CAM) resistance to ensure healthy growth until the OD600 was around 0.6, which correlates to log phase growth. Next, aliquots of these cells were "washed" in either pH 6, 7, 8, or 9 M9 media to create a 1:3 dilution. This washing step ensured no pH alteration occurred due to previous unaltered media. The culture is grown overnight in the pH media with CAM resistance. The next day, aliquots of those cultures were placed in a 96 well plate reader to calculate relative fluorescence values using the 455 nm excitation of the YGCP. Fluorescence data was normalized using OD600 values. These results are in figure 1. | ||
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https://static.igem.org/mediawiki/2016/a/ad/T--Austin_UTexas--pH_Dependent_Promoter.jpeg | https://static.igem.org/mediawiki/2016/a/ad/T--Austin_UTexas--pH_Dependent_Promoter.jpeg | ||
Revision as of 19:26, 25 October 2016
CpxR binding site attached to a yellow-green color protein (YGCP) acts as a neutral pH indicator.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
CpxA-CpxR is a two-component mechanism that is activated at pH 7.4 and repressed at pH 6.0. CpxA is an intermembrane protein that autophosphorylates at a certain external pH, CpxR (a kinase) then gets phosphorylated by CpxA and acts as a transcription factor for the downstream gene, YGCP in this case. This system originally is a transcription factor for the virF gene, but virF was replaced with a reporter. The original sequence was found in Shigella sonnei, but E. coli has a homolog of these proteins[1] [2]. Only the appropriate BioBrick prefix/suffix and CpxR binding site were required to design the part. Used BBa_K2097002 as the control series.
[1] Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528
[2]Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069
Experimental Design
Top10 E. coli are grown in unaltered M9 minimal media with chloremphenicol (CAM) resistance to ensure healthy growth until the OD600 was around 0.6, which correlates to log phase growth. Next, aliquots of these cells were "washed" in either pH 6, 7, 8, or 9 M9 media to create a 1:3 dilution. This washing step ensured no pH alteration occurred due to previous unaltered media. The culture is grown overnight in the pH media with CAM resistance. The next day, aliquots of those cultures were placed in a 96 well plate reader to calculate relative fluorescence values using the 455 nm excitation of the YGCP. Fluorescence data was normalized using OD600 values. These results are in figure 1.
Figure 1.The blue data points represent fluorescent readings with the control series (BBa_K2097002), while the orange data points are the Cpx construct. As one can see, the Cpx construct shows increased fluorescence as the pH increases from 6 to 9.