Difference between revisions of "Part:BBa K118011:Experience"

SDU-Denmark

Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655ΔcyaA in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD₆₀₀measurements. Graph shows intensities of mRNA-gfp normalized according to intensity of 5S.

We set up to measure promotor activity of PcstA by measurering levels of gfp-mRNA with bacteria transformed with PcstA-gfp (BBa_K1135002). MG1655ΔcyaA, LB was used as a negative control. Samples were collected at different OD₆₀₀-measurements. A single sample from the negative control was collected at OD₆₀₀ = 0.3. The RNA from the samples was purified and a Northen Blot with gfp and 5S probes was performed.

Generally during the exponential phase of the bacteria, they have a high level of transcriptional activity. However, levels of 5S rRNA are relatively constant at all times. The transcription of gfp increase as the cells enter exponential phase between the two OD₆₀₀ measurements 0.1 and 0.3. As expected, very low levels of gfp can be detected in the negative control. This strain lacks the ability to generate cAMP, and thus very little transcription is induced. The small amounts of gfp could be explained by leakiness of PcstA or that CAP alone initiates some transcription.

In the setup with WT, LB it is quite clear that the amount of gfp rises, compared to WT, LB+0.2% glucose. Transcription is clearly affected by the presence of glucose. One measurement WT, LB OD₆₀₀ = 0.8 stands out. The result is not readily explained, but is probably due to some error. But the tendency of the results correlates with the knowledge of the invert relationship between glucose and cAMP. Glucose signaling will repress adenylate cyclase-activity, thus intracellular levels of cAMP will be low in high-energy states, and little transcription of gfp will be initiated.

BBa_K118011 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 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]

BBa_K118011 2 Not understood WHU-China 2012

Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose. What's more, we have constructed an indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.

The indirect regulatory pathway

In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration. In another word, the indirect pathway can be activated by glucose

For more information, please go to wiki of WHU-China 2012 on [http://2012.igem.org/Team:WHU-China/Project?catalog=2#Indirect_Pathway_Design Indirect pathway]

BBa_K118011 3 Not understood UCL_PG 2013

UCL_PG 2013 aka. SPECTRA

pCSTA.RBS.mKeima.TT is compared with the preceding biobricks. We actually reconstructed pCSTA.RBS.eGFP.TT and pCSTA.RBS.mRFP.TT from 2013 distribution to test the modularity of the generator bricks. They are all successfully clone in few days.
During the comparison, we used a low volume of living E.coli for fluorescent detection. They are not lysed. The purpose is to demonstrate suitability of each fluorescent protein in tiny scale kinetic study. Please refer to our project page for more!

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