Latest revision as of 03:37, 22 October 2021

Mutated Ribose Binding Protein (RBP) to bind BTCA

This protein was computatinal designed by the IGEM 2021 Bielefeld Team to have a better affinity to Benzenetricarboxylic acid. It is used to iniate a signalling cascade.

Usage and Biology

In our project, we created a plant-based detection system for degradation products of chemical weapons, for which a functional and specific receptor is crucial. We computationally designed a receptor based on a ribose binding protein to bind a given chemical. For the design we utilized Rosetta in combination with EvoDock, a Python script that adds an evolutionary approach, and increased the efficiency of the design process. We experimentally demonstrated the binding of Benzenetricarboxylic acid (BTCA) by the computationally engineered receptor and thereby verified the functionality of our model. This was performed in two approaches, in vitro binding analysis and in vivo by the activation of a signaling cascade resulting in the induced expression of GFP upon specific ligand binding in bacteria. Our design pipeline can be re-applied to engineer receptors for countless applications. Following our detailed workflow descriptions, future iGEM teams are able to design their own specific receptors as well as binding proteins.

Overexpression of computationally designed receptors

After the successful implementation of the experiments towards establishing the signaling cascade with RBP overexpression, we decided to overexpress our computationally designed diisopropyl methylphosphonate (DIMP) and 1,3,5-benzenetricarboxylic acid (BTCA) receptors for the purpose of testing them in-vivo. We used vectors pRSETB-DIMP-receptor/BTCA-receptor for expression of the receptors for in-vitro testing, were used for a co-transformation with the signaling cascade in BL21(DE3). The above-mentioned plasmids belong to the same ori incompatibility group, but are sustained by the cell by the same mechanisms as for the pJOE-RBP construct used in co-transformation. After heat shock transformation, colonies were picked and cultivated in 15 mL LB-medium supplemented with ampicillin and chloramphenicol, and grown under the same conditions as before. After plasmid isolation, the correct incorporation of the parts of the signaling cascade and the respective receptors were verified by PCR. A growth experiment was conducted. Cells were grown in 1 mL LB supplied with antibiotics in a 96-deep well plates at 37°C on a rotary shaker at 500 rpm. Additives were added in the same manner as before, combinations of additives are shown in the table below. Three biological replicates were tested. Fluorescence was measured in 96-well plates in the Tecan-reader. For each well, nine measurements were conducted, and cells were diluted to an OD of 1. The fluorescence of the LB-medium was subtracted.

All three replicates show the same trends in fluorescence levels. The sole addition of either arabinose or BTCA in various concentrations led to fluorescence levels similar or below the fluorescence levels of the negative control (Figure 7). This finding indicates no significant activation of the signaling cascade. Addition of arabinose and BTCA led to significantly higher fluorescence levels, with the culture exposed to the highest concentration demonstrated the highest fluorescence. Addition of 1% ethanol to the cultures grown on arabinose, also increased fluorescence in comparison to the negative control. This can be explained by the osmotic pressure from ethanol, which activates the osmotic stress responsive promotor. However, the addition of the ligand only did not result in higher fluorescence and the arabinose supplementation is not sufficient for inducing osmotic stress, it is likely, that the signaling cascade is the main contributor to the higher fluorescence levels. This provides evidence for the receptor binding the ligand. This conclusion is in line with the results of the in-vitro testing and is a proof of concept for the computational receptor design of the receptor binding specifically to the BTCA. To provide further evidence, yet another growth experiment was done to compare the abilities of the BTCA-receptor and RBP to activate the signaling cascade. The DIMP-receptor showed no affinity to DIMP nor ribose (data not shown). Possible ways to obtain a functional DIMP-receptor are computational re-design, or mutation of RBP by Darwin assembly. Our attempt toward construction thereof can be found in description of our library experiments.

Endogenous receptor vs. computationally designed receptor

For the testing of efficiency of ligand binding of the RBP compared to the computationally designed BTCA-receptor, we conducted a cultivation experiment under the same conditions as the last experiments. The combinations of additives are shown in the table 4. Biological triplicates of the BTCA-receptor overexpressing strain 2 and of a RBP overexpressing strain were used. Fluorescence was measured in 96-well plates in the Tecan-reader. Each well was measured nine times, cells were diluted to an OD of 1. The fluorescence of the background of LB-medium was subtracted.

The ethanol, and negative controls do not differ significantly, confirming that ethanol has no effect on the fluorescence, and that in fact BTCA activating the signaling cascade is the main contributor to the increased fluorescence (Figure 8). The RBP overexpressing culture supplemented with ribose shows no significantly higher fluorescence than the negative control. One possible reason for this is, that no arabinose was added to the culture media. Further in-depth experiment to elucidate the exact reason were not concluded yet due to time constraints, however the most important comparison between cultures/constructs with overexpressed RBP and the BTCA-receptor with addition of BTCA are currently undergoing. The mean fluorescence intensity measured is approximately twice as high for the BTCA-receptor binding BTCA as for RBP binding BTCA. A one-sided T-test for unpaired data showed a significant higher fluorescence for the BTCA-receptor binding BTCA (p = 0.041, n = 3). This shows that the BTCA-receptor has a higher affinity to BTCA than RBP. It is a proof of concept for the computational receptor design and our project.

3D deconvolution widefield fluorescent microscopy and super resolution 3D structured illumination microscopy

Lastly, we performed microscopy experiments to detect the GFP induced by the signaling cascade on a single cell level. For this purpose, we had access to a Deltavision OMX V4, a cutting-edge life cell imaging microscope, which uses Fourier transformation to gain a resolution higher than the diffraction limit of visible light. We did 3D deconvolution Widefield Fluorescent Microscopy and super resolution 3D-structured illumination microscopy. The BTCA-receptor culture supplied with BTCA and the respective negative control were used in this experiment. Figure 9 shows the 3D deconvolution widefield fluorescent microscopy, the differential interference contrast channel and the eGFP channel and the merged overlay of both channels in various magnifications. Evidently, the BTCA supplemented BTCA-receptor culture displayed a GFP fluorescence while the negative control showed none. The results of the super resolution 3D-structured illumination microscopy can be seen in figure 10. The differential interference contrast channel, the eGFP channel, and the merged overlay of both channels in different magnifications are shown. The super resolution 3D-structured illumination microscopy confirms once again that we obtain a green fluorescence after arabinose induction and addition of BTCA to the culture, while the negative control showed no fluorescence.

Sequence and Features