Part:BBa_K2398555

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 regulatory elements that are used for the binding of specific molecules to release an intrinsic RBS which subsequently facilitates the translation of the downstream gene of interest. This riboswitch, in particular, was designed in silico by our MAWS 2.0 software for the specific binding of one organosilicon. It binds the product (5) that is depicted in Fig. 1 and makes the transcript of the reporter accessible for ribosome binding. The proof-of-concept for this riboswitch has been successfully demonstrated in a riboswitch binding assay. As a next step, this part can be implemented in the directed evolution approach of phage-assisted continuous evolution (PACE) or in the phage-related discontinuous evolution (PREDCEL) approach. Therein, it aims to improve the organosilicon synthesis by regulating the evolutionary selection pressure via gene III expression.

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]