Team:SDU-Denmark/Tour51
"It is beyond a doubt that all our knowledge begins with experience." - Immanuel Kant
Reporter System
We sat out to improve the original reporter system – the PcstA induced transcription of the gene encoding Red Fluorescent Protein (RFP). We wanted to further characterize the PcstA promotor, part: BBa_K118011.
PcstA is a carbohydrate stress induced promotor. In absence of glucose the enzyme adenylate cyclase will synthesize cAMP from ATP. cAMP will bind CAP CAPCatabolite activated protein, en-coded by crp. and the complex recruit RNA polymerase to the promotor. There is an inverse relationship between cAMP and glucose, and this means that glucose can repress promotor activity.
Originally our reporter system was based on the part BBa_K861173 in which the promotor controls transcription of rfp. However it is challenging working with RFP, especially due to its long folding time. Therefore we decided to perform the experiments with another brick, which also contained PcstA; BBa_K1135002. In this brick PcstA will initiate transcription of the gene encoding GFP.
K1135002 was transformed into wildtype (WT) MG1655 and MG1655ΔcyaA as a negative control. From an overnight culture, three setups was prepared; a culture in regular LB medium, a culture in LB medium + 0.2% glucose, and ΔcyaA in LB medium. 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.
We also tried to measure RNA levels induced by cAMP. This experiment was performed in two adenylate cyclase deficient strains; MG1655ΔcyaA and BTH101. In this setup, 1 mM cAMP was added to the medium of exponential cells and samples were collected at specific times. However, levels of RNA did not seem to be affected by cAMP. But this is probably not due to insensitivity of PcstA to cAMP. The most reasonable explanation is that cAMP failed to activate transcription of GFP, because the molecule couldn’t pass the cell membrane.