Team:Thessaloniki/Improve

Part Improvement

Part Improvement

We submitted BBa_K2839017 as an improvement over BBa_K784005, a theophylline riboswitch designed by iGEM12_Technion Team.

Theophylline Riboswitches can be used for the conditional control of gene expression. In the absence of theophylline, the RNA transcript coding for the fluorescent marker forms a stem loop that limits the access of ribosomes to the ribosome binding site (RBS), thus inhibiting translation initiation. With the addition of theophylline this loop undergoes a conformational change, allowing the binding of the ribosome at the RBS and permitting translation initiation. As, the main focus of our project is the precise control of gene expression, we implemented a Theophylline Riboswitch in the TAL Effector stabilization system, granting inducibility. Therefore, we achieve theophylline-dependent gene expression , decoupled from copy number.

According to the experimental data submitted by iGEM12_Technion Team, the theophylline riboswitch they used did not respond to changes in theophylline concentration. We concluded that the lack of functionality possibly stemmed from a disturbance in the riboswitch’s conformation. We wanted to investigate whether mCherry coding sequence, which was the reporter that Team Technion used, affected the riboswitch’s secondary structure. Therefore, in order to restore it’s function, we initially replaced the mCherry fluorescent marker with sfGFP. From the flow cytometry measurements,we observed no fluorescence and thus no functionality. As a second approach, we used sfGFP protein fused with the first 99 nucleotides of luciferase, as we believed it i not interact with the aptamer sequence.

Results

Fluorescence intensity measurements were conducted in DH5a E.coli cells, following this flow cytometry protocol.

The BBa_K2839017 (12.1 Theophylline Riboswitch +sfGFP-luciferase fusion protein) and BBa_K784005 expressing sfGFP were inserted in pSB1C3 backbone, in order to determine their functionality. To examine whether activation of sfGFP expression was dose-dependent, cells were treated with 4 different Theophylline Concentrations: 0.5 mM, 1 mM, 2mM and 8mM. The results indicated that higher doses of Theophylline lead to higher fluorescence levels in the BBa_K2839017 riboswitch, which uses sfGFP-luciferase fusion protein as a reporter. On the contrary, BBa_K784005 did not exhibit any fluorescence in the Theophylline concentrations tested.

Performance of 12.1 riboswitches when different theophylline concentrations were added.

We restored BBa_K784005 functionality using N-terminal Fusion sfGFP with the first 99 nucleotides of luciferase. The 99 nucleotides of luciferase can be fused with proteins that remain functional upon N-terminal addition,ensuring the riboswitch’s proper function.