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Applications of BBa_K1135002
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UNIQd90765b1986e284f-partinfo-00000000-QINU
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SDU-Denmark
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 Transcriptional activity of PcstA during growth by measurering RNA. Negative control of MG1655Δ cyaA in LB, WT in LB+0.2% glucose, and WT in LB. Samples collected at different OD 600measurements. 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.
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UNIQd90765b1986e284f-partinfo-00000003-QINU
UNIQd90765b1986e284f-partinfo-00000004-QINU
