"It is beyond a doubt that all our knowledge begins with experience." - Immanuel Kant

Characterization of PcstA

Figure 1: The experimental setup

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 promoter PcstA PromoterPcstA is a carbon stress induced promotor. In absence of glucose the enzyme adenylate cyclase will synthesize cAMP from ATP. cAMP will bind CAP, catabolite activator protein, encoded 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., part: BBa_K118011, by measuring the levels of mRNA through a Northern Blot.

Originally our reporter system was based on the part BBa_K861173 in which the promotor controls transcription of rfp. However, it is challenging to work with RFP, partly due to its relatively 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.

Figure 2: Transcriptional activity of PcstA during growth measured by RNA levels.

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 increases 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.

gfp-mRNA from ΔcyaA was slightly increased after 8 minutes. This is not as expected, but could be possibly be due to the fact that cAMP failed to pass the cell membrane. ">

Figure 1: Northern blot of gfp-mRNA induced by cAMP

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 subsequently 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>/i>, because the molecule were unable pass the cell membrane. However, RNA levels were at best slightly increased after approximately 8 minutes (see figure 2). To study the direct effect of cAMP on the promotor, one must use a different approach.