"I've always believed that if you put in the work, the results will come." - Michael Jordan

Results

Figure 1: Transcriptional activity of PcstA during growth measured by RNA levels.

In the following chapter you will see all our results from the lab.

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Figure 2: Streaks of BTH101 on LB/X-gal plates, containing pSB1C3-T18 + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25, pSB1C3-T18-Zip + pSB1K3-T25 and pSB1C3-T18-Zip + pSB1K3-T25-Zip.

Our goal was to screen for peptide aptamers using a bacteria-based system. This system can be used to study protein-protein interactions. We used the bacterial two-hybrid system which is based on the reconstitution of the adenylate cyclase. In order to detect the rise in intracellular cAMP we used a Red Fluorescence Protein (RFP) reporter system with a cAMP-activated promoter, PcstA. Validation of a functional two-hybrid system and characterization of the reporter system was needed, before we could screen for peptide aptamers.

Characterization of promotor PcstA, was done by meassuring levels of mRNA during growth. The promoter is sensitive to the glucose concentrations, which was tested in the experiment.

The bacterial two-hybrid system has never been introduced to iGEM before. For this reason we wanted to validate the function of the system. To examine if the bacterial two-hybrid system can be used to study protein-protein interactions, we made a control experimental setup, where the leucine zipper region from the GCN4 yeast (Saccharomyces cerevisiae) protein were fused to the T18 and T25 domains (T18-Zip+T25-Zip). As leucine zippers form homodimers, their interaction should lead to functional complementation between T18 and T25.

We used the E. coli K12 BTH101-strain, which is deficient in the gene encoding adenylate cyclase, cyaA. Our results showed that only when leucine zippers was fused to both T18 and T25, complementation was observed. We wanted to carry out a screening for peptide aptamers, but due to delayed deliverance of our nucleotide library and problems inserting it in our scaffold, we did not accomplish this.

17 parts were sent to the Parts Registry. In Submitted Parts you can browse through our parts and be redirected to the Parts Registry. Here you will find more information on our parts, e.g. function, sequencing and characterization.

This year we participated in iGEM's Second International InterLab Measurement Study. The goal of this study was to measure fluorescence from three different devices expressing GFP. We were able to measure the fluorescence of these devices using FACS. The data we acquired showed that there was a relative difference in promoter activity in the mutated promoters compared to the wild type.