Difference between revisions of "Team:Heidelberg/Organosilicons"

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{{Heidelberg/templateus/Imagebox|https://static.igem.org/mediawiki/2017/4/40/T--Heidelberg--PlateAssay.png|Figure 3:|The riboswitch binding assay in a 96-well plate immediately after substrate addition. Rows show biological triplicates (n = 3) and riboswitch activators are compound (3) ethyl 2-(dimethyl(phenyl)silyl)propanoate and compound (1) dimethyl(phenyl)silane.|}}
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{{Heidelberg/templateus/Imagebox|https://static.igem.org/mediawiki/2017/4/40/T--Heidelberg--PlateAssay.png|Figure 3:|The riboswitch binding assay in a 96-well plate immediately after substrate addition. Rows show biological triplicates (n = 3) and riboswitch activators are compound (3) ethyl 2-(dimethyl(phenyl)silyl)propanoate and compound (1) dimethyl(phenyl)silane.|}}
  
 
             The purified construct was retransformed into DH10beta and incubated at 37°C overnight. To carry out the assay in biological triplicates, three clones were picked and inoculated separately overnight in 5ml LB including chloramphenicol.
 
             The purified construct was retransformed into DH10beta and incubated at 37°C overnight. To carry out the assay in biological triplicates, three clones were picked and inoculated separately overnight in 5ml LB including chloramphenicol.

Revision as of 16:06, 1 November 2017


Organosilicons
Synthesis of organosilicons and cytochrome engineering
Organosilicons, compounds containing bonds between silicon and carbon, provide completely new structural moieties with altered properties and metabolism. By utilizing a well-known and previously engineered cytochrome cFranz.2013 as a catalyst, it is conceivable to synthesize carbon-silicon compounds applicable for medical treatment and agriculture applications e.g. in Alzheimer’s disease or as insecticides, respectively. We tended to generate novel organosilicon-forming enzymes by evolving enhanced cytochrome c variants by using a phage-assisted continuous evolution (PACE) approach. In a stepwise proof-of-principle design, we first show the production of two different organosilicons analyzed via the GC-MS method, and second, the viability of a riboswitch-coupled reporter system, which detects one of the most valuable compounds derived from Organosilicon formation. This proof-of-principle is a step further towards biocatalysts, which are highly efficient, environmentally friendly, and will greatly contribute to the production of novel carbon-silicon bonds.

Introduction

Organosilicons are organometallic compounds that consist of carbon-silicon bonds. They are comparable to their corresponding organic analogs but differ in their intrinsic properties. These differences, especially the chemical properties of silicon and its bond formation tendencies, have a significant impact on their bioavailability and their application in medicineFranz.2013. Recent publications cluster their unique features into three categories: The first category comprises the chemical properties of silicon bonds. Typically, silicon forms longer bonds at different angles, which leads to diverse ring conformations and thus, alterations in reactivity. Furthermore, its preference to form single bonds leads to chemical compounds that have a higher intrinsic stability than their carbon analogs. The second category represents the bioavailability of organosilicons. They are more likely to overcome the membrane barrier of cells as they are more lipophilic compared to their respective carbon counterparts. The third - and most important - category deals with the medical application of these compounds. Due to their aforementioned tendency to form single rather than double or triple bonds, they display a viable source for stable pharmaceuticals, which are inaccessible as carbon-based molecules. Additionally, the more electropositive nature of silicon facilitates hydrogen bond formation and conveniently increases the acidity of the compounds. As a result, organosilicons address the major issue in the synthesis of bioactive pharmaceuticals, the design of pro-drugs, as well as a safe medicine with a genuine biomedical benefit. Thus, their main advantage is to operate as pro-drugs due to their thermodynamic stability, but aqueous and acidic instability. On top, as we know so far, silicon is nonhazardous by itself, which makes it a valuable source for further biomedical research.

Our Idea

As a proof-of-principle, we wanted to show and harness the potential of organosilicon-forming proteins. Therefore, we used a previously engineered cytochrome c enzyme and coupled organosilicon-production directly to a reporter expression. Thereby, we were focusing on a small molecule-sensing riboswitch as proposed underlying mechanismHenkin.2008. This riboswitch was designed in silico using the MAWS 2.0 software, that was provided by the iGEM Team Heidelberg 2015. In a step-by-step approach, we wanted to produce an organosilicon which, in the end, could be tested with the designed riboswitch to express the NanoLuc reporter (Promega). The NanoLuc is currently the most sensitive luciferase available, and is able to show us a significant output despite using only a small amount of substrate.
Figure 1:
Our workflow for organosilicon production and cytochrome engineering.
Figure 2:
3D structure of the cytochrome c derived from Rhodotermus marinus (PDB) that is used as the catalytic unit in the production of organosilicon. Depicted as yellow sticks is the heme prosthetic group of the electron carrier protein. The protein part of the cytochrome c is illustrated as orange ribbon structure.

Experimental procedures

Design and cloning of the riboswitch and cytochrome c constructs

The educts for the organosilicon synthesis were commercially available in the case of compound dimethyl(phenyl)silane (2) and ethyl 2-diazopropanoate (5) or were custom synthesized by Fabian Ebner (Greb group, ACI Heidelberg, Germany) in the case of compound 4-(dimethylsilyl)aniline (1). The corresponding riboswitch was designed accordingly using the MAWS 2.0 software developed by the iGEM Team Heidelberg 2015. To obtain the riboswitch sequence, the chemical structure of the desired product was in silico aligned to randomly generated RNA sequences which were scored according to their ability to form hydrogen bonds with the product. The most favorable sequence (BBa_K2398555) was ordered as oligos which were annealed in a single-cycle Touch-Down PCR, diminishing the temperature by 0.1°C x sec-1 95°C to 10°C. The sequence was ordered as oligos and not as gBlock to ensure overhangs of a specific length and sequence at the 5’ and 3’ ends. The vector and reporter were amplified via PCR and purified by gel extraction (Qiagen). The final plasmid was assembled by using an equimolar concentration of the vector, reporter, and the riboswitch in a golden gate reaction. The plasmid was amplified after transformation in DH10beta cells and purified via plasmid purification (Qiagen). To make organosilicon production more approachable 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 desired best basic part (BBa_K2398000). For cloning purposes, we ordered the optimized version as gBlock containing a biobrick prefix and suffix for restriction cloning. We were using this part as a triple mutant, created by F. ArnoldKan.2016, that showed a higher activity towards our substrates.

Riboswitch binding assay

Figure 3:
The riboswitch binding assay in a 96-well plate immediately after substrate addition. Rows show biological triplicates (n = 3) and riboswitch activators are compound (3) ethyl 2-(dimethyl(phenyl)silyl)propanoate and compound (1) dimethyl(phenyl)silane.
The purified construct was retransformed into DH10beta and incubated at 37°C overnight. To carry out the assay in biological triplicates, three clones were picked and inoculated separately overnight in 5ml LB including chloramphenicol. The assay reaction was performed in a transparent 96-well plate (Greiner) with a final volume of 250µl per well (fig. 3). The light absorbance was measured at 460nm. The background absorbance was determined by measuring separately medium only, medium including cells, and medium including cells and the Nano-Glo substrate. The cell concentration was adjusted to a final OD600nm of 0.6 per well. Following, the riboswitch activity was detected in the wells that additionally contained either the educt (2) as the initial substrate or the product (3) to which it was specifically designed. Therefore, the reaction efficiency was tested for the compounds (2) and (3) using either 5mM or 15mM concentrations, respectively. The reaction mixtures were incubated at RT for 10min without the Nano-Glo substrate. After the incubation, the Nano-Glo substrate was added fresh to each well (125µl) to ensure its maximum reactivity. The light absorbance was immediately measured using the Tecan Infinite M200 Pro plate reader.

Results

Synthesis of the organosilicon compounds

Following the successful synthesis of the two compounds (3) and (4), they were subjected to gas chromatography-mass spectrometry (GC-MS) for the analysis of validation and calculation of the conversion rate. Figure 4 shows the ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate product with a retention time corresponding to 11.7 minutes. The silicon educt 4-(dimethylsilyl)aniline that emerges at 6.9 minutes implies that there is no complete conversion, possibly due to enzyme inactivity or an unfavorable reaction. Indeed, the conversion rate is 47.5%, which differs from the literature value of 70%Kan.2016. Nonetheless, the amount of the product formed was sufficient for all further experiments. The unconverted diazo educt ethyl 2-diazo propanoate corresponds to the retention time peaks at 7.2 and 7.4 minutes. The small peak at 10.8 minutes is likely a side product of the reaction. The successful product synthesis was further confirmed by mass spectrometry. Fig. 5 shows the mass spectrum of the breakdown of the product and its correct mass of 251 Dalton.
Figure 4:
Gas chromatogram for the reaction of educt (1) and (5) to the product (3). 11.7 minutes retention time, indicates product formation. Unconverted educts converge 6.9 and 7.2, 7.4 minutes
Figure 5:
Mass chromatogram shows the breakdown of the product (3) ethyl 2-((4-aminophenyl)dimethylsilyl)propanoate. The product itself corresponds to a mass of 251 dalton
The GC-MS analysis of the organosilicon ethyl 2-(dimethyl(phenyl)silyl)propanoate is demonstrated in Fig. 6 and 7. Hereby, Fig. 6 shows the complete conversion of the silicon educt dimethyl(phenyl)silane. The product emerges at a retention time of 9.2 minutes. With >99% conversion rate the product was obtained in high quantity. As described before, the MS analysis verified the product and its correct mass of 236 Dalton (Fig. 7).
Figure 6:
Gas chromatogram for the reaction of educt (2) and (5) to the product (4). 9.2 minutes retention time indicates product formation.
Figure 7:
Mass chromatogram shows the breakdown of the product (4) ethyl 2-(dimethyl(phenyl)silyl)propanoate. The product itself corresponds to a mass of 236 dalton
The reaction without the enzyme present served as negative control and is depicted in Fig. 8. As expected, only the two educts emerged after their respective retention time and no product was obtained.
Figure 8:
Gas chromatogram for the control reaction where the enzyme has been substituted.
Conclusively, satisfactory concentrations of the products could be achieved and, in case of the organosilicon (3), it could be purified to >98% chemical purity.
The emission rates detected at 460nm were plotted as indicated in Fig. 9. The conditions of each reaction are specified below their respective bars. All measurements are already subtracted for the background emission. Interestingly, the addition of 15 mM of the compound (3) resulted in a higher emission rate compared to the sample containing the 5 mM concentration. As the compound (3) was specifically designed for the riboswitch it was striking to detect a significantly higher emission rate compared to the original precursor compound (1), indicating a higher binding affinity. Notably, these results were very reproducible along biological triplicates, as suggested by the small error bars. The increase in enzyme activity upon addition of the specific riboswitch activator was determined in relation to the enzyme activity in presence of the initial substrate. This is demonstrated in Fig. 10 that shows the 1.5 fold enzyme activity when using the newly synthesized substrate.
Figure 9:
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 10:
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.

Outlook

Figure 11:
The selection of any small molecule synthesis is implemented into the product formation drive PREDCEL approach. The selection pressure is applied by the most efficient riboswitch binding and subsequent gene expression activation.
Overall, these results show the proof-of-principle of the application of organosilicons in the use of riboswitches. Moreover, it presents the promising opportunity to further subject small molecule-sensing riboswitches to an enzyme PACE approach (PREDCEL) for the enhancement of downstream reactions such as gene III activity to facilitate phage propagation (Fig. 11). This means, that the formation of the desired product is coupled to a riboswitch-mediated positive and negative selection approach. All riboswitch sequences were designed in silico using the Making Aptamers Without SELEX (MAWS) 2.0 software. A pool of preselected enzyme isoforms generated by the software tool AiGEM manufactures a wide variety of products. In the case of the positive selection, the desired product binds to the riboswitch that was specifically designed to be activated by it. The subsequent conformational change then facilitates phage propagation via the enhanced expression of the gene III. Thus, the enzyme that most efficiently forms the product of interest is enriched over time in a positive selection process. To ensure that enzymes that synthesize undesired products are not propagated, negative selection is conducted. Thereby, different riboswitches mediate the negative gene III expression upon product binding. The negative gene III inhibits the further phage propagation and thus, decreases the abundance of all enzymes not specifically forming the desired productCarlson.2014. Therefore, under the evolutionary pressure of phage propagation, the selection is driven towards the enzyme of interest.

References