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

Results

In the following chapter you can see the lab-results that we have accomplished during the last couple of months. We have been working hard, and blood, sweat and tears have brought us the data that we can present to you in the next couple of pages.

Our goal was to make a bacteria-based system that can be used to study protein-protein interactions for screening of peptide aptamers. We used the bacterial two-hybrid system that is based on the reconstitution of the adenylate cyclase. To detect the rise in intracellular cAMP we used a RFP reporter system with a cAMP-activated promoter, PcstA. Before we could make a screening for peptide aptamers we wanted to characterize the RFP reporter system, and validate the functionality of the bacterial two-hybrid system.

PcstA-based reporter System
To improve characterization of the carbon stress promotor PcstA, we measured levels of mRNA which it induced transcription of during growth.

Two-Hybrid System

To validate if the main components of the "Two-hybrid System", T18 and T25 can be used to study protein-protein interactions, we made a control experiment, where the leucine zipper region from the GCN4 yeast (Saccharomyces cerevisiae) protein was 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. 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.

We used the BTH101-strain, which is an Escherichia coli K12-strain, deficient in its gene encoding adenylate cyclase, cyaA. Our results showed that only when leucine zippers was fused to both T18 and T25, complementation was indeed observed.

Submitted Parts
After a lot of PCR, digestions, ligations, transformations, colony-PCR and mini-preps, we ended up making 17 parts that were send to the parts registry. In the page “Submitted Parts” you can browse through our parts and from there be directed to the parts registry where you can find more information on our parts, e.g. function, sequencing and characterization.

InterLab Study
This year we also participated in the Second Internation InterLab Measurement Study. The goal for 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.

Dig deeper to get a more detailed description of our results.