IntroductionOrganosilicons are organometallic compounds that contain carbon-silicon bonds. In comparison to their respective organic analogs, they display different intrinsic properties due to the distinct chemical properties of silicon. The bond formation tendencies of silicon have a significant impact on their bioavailability and their applicability in pharmacy
Recent publications cluster their unique features into three categories
The second category represents the bioavailability of organosilicons
Recently, a cytochrome c variant was described, which is able to catalyze the formation of C-Si bonds
Our IdeaAccording to our idea to evolve proteins by PACE and PREDCEL, we also envisioned to further develop the previously engineered cytochrome c. Therefore, we linked organosilicon-production directly to a reporter expression via a small molecule-sensing riboswitch
Design and cloning of the riboswitch and cytochrome c constructsThe educts for the organosilicon synthesis were commercially available in the case of dimethyl(phenyl)silane (1) and ethyl 2-diazopropanoate (3) or were custom synthesized by Fabian Ebner (Greb group, ACI Heidelberg, Germany) in the case of 4-(dimethylsilyl)aniline (2) (Fig. 4). The corresponding riboswitch was designed accordingly using the MAWS 2.0 software developed by the iGEM Team Heidelberg 2015. The most favorable sequence (BBa_K2398555) was ordered as oligos, which were annealed in a single-cycle Touch-Down PCR, decreasing the temperature by 0.1°C x sec-1 from 95°C to 10°C. The sequence was ordered as oligos and not as gBlock to ensure overhangs of a specific length at the 5’ and 3’ ends. Vector and reporter were amplified via PCR and purified by gel extraction (Qiagen). The final plasmid was assembled by using equimolar concentrations of vector, reporter, and the riboswitch in a golden gate reaction. The plasmid was transformed into DH10beta cells and purified by plasmid purification (Qiagen).
To make organosilicon production more accessible for other iGEM Teams, we codon optimized the wild-type cytochrome c derived from Rhodotermus marinus and cloned it into the pSB1C3 vector. We are proud to present you this part as our best basic part (BBa_K2398000). For our purpose, we used a triple mutant created by F. Arnold
Riboswitch binding assay
Synthesis of the organosilicon compounds
The GC-MS analysis of the second organosilicon ethyl 2-(dimethyl(phenyl)silyl)propanoate is demonstrated in the Fig. 5 and 6. The educt dimethyl(phenyl)silane was completely converted, and the product emerges at a retention time of 9.2 minutes (Fig. 7). The mass spectrometry analysis verified the product with a mass of 236 daltons (Fig. 8). The reaction without the enzyme represents our negative control and is depicted in Fig. 9. As expected, only the two educts emerged after their respective retention time.
In summary, sufficient concentrations of the organosilicon products were synthesized, and in case of the organosilicon (5) also purified to >98% chemical purity.