Difference between revisions of "Part:BBa K2398555"

Revision as of 20:27, 1 November 2017

Organosilicon binding Riboswitch

This in silico designed riboswitch by the MAWS software provided by the iGEM Team Heidelberg 2015 is made for the binding of Organosilicons. To be precise, ethyl 2-(dimethyl(phenyl)silyl)propanoate was used as the substrate. It can be used to 2.5-fold activate your downstream gene of interest as it is shown by our experiments. This part was ordered from IDT as oligo and cloned in pSB1C3 containing NLuc (Promega) as a reporter gene. As riboswitches are only specific for one substrate this part is only validated for the use of the appropriate substrate.

Usage and Biology

Riboswitches, in general, are used for the binding of specific molecules to release an intrinsic RBS that leads to translation of the downstream gene of interest.

Characterization

To prove that our riboswitch can activate a downstream localized gene of interest we designed a reporter system in which we cloned the in silico designed riboswitch behind a promoter region and in front of the luciferase NLuc, purchased from Promega.

Figure 1: Used compounds for the characterization of the organosilicon-sensing riboswitch

Figure 2: Light emission detection of the NanoLuc reaction for different riboswitch activators and concentrations. Addition of compound (3) to the reaction resulted in increased enzyme activity as indicated by the two bars on the left-hand side compared to the compound (1) reaction.

Figure 3: Fold enzyme activity upon addition of compound (3) compared to compound (1) at either 5 mM concentration (left bar) or 15 mM concentration (right bar). At both concentrations, compound (3) exceeded the reaction activity of compound (1) by 1.5 fold.

Sequence and Features