Team:BOKU-Vienna/Improve

Navbar

Project Inspiration Description

Theophylline_Riboswitch (transcriptional) BBa_K3015004

The iGEM Team BOKU-Vienna improved the part BBa_K784005 by designing a Theophylline riboswitch that increases expression of downstream genes while offering multiple advantages.
The riboswitch does not need to be scarlessly fused to the gene of interest. Our riboswitch can be combined with a promoter, RBS or gene of interest by RFC10 or TypeIIS compatible cloning. Also, the Theophylline riboswitch works on a transcriptional level, meaning the intrinsic termination design stops the gene expression during the transcription. The aptamer sequence will form a strong secondary structure to the terminator sequence to form a hairpin structure with the spacer sequence building a loop (see figure 1). This loop binds to the nusA protein on the RNA-polymerase and stops transcription. Due to weak binding between the RNA-polymerase and the Uracil-stretch, the polymerase will dissociate and terminate transcription. That way RNA sequences can be used as a target.

The Theophylline riboswitch shows a 1.5 fold increase in the presence of 10µM Theophylline, a 4 fold increase of gene expression at 100µM Theophylline and a 6.4 fold increase at 1mM Theophylline (see figure 2).

See part BBa_K3015007 for more information on the measurement and experiment.

Figure 1: Theophylline aptamer with intrinsic termination secondary structure

Figure 2: Relative fold increase at different Theophylline concentrations

T7 RNA Polymerase (No BbsI/BpiI) BBa_ K3015006

Since BbsI/BpiI can be a very important restriction enzyme for golden gate cloning in some labs, we decided to remove those recognition sites by codon swapping from the Part BBa_K14001 to improve the applicability.