<B>Figure 6. FPLC C2 protein purification</B>

<B>Figure 6. FPLC C2 protein purification</B>

(A) Polystyrene-based Ni-NTA column (Nuvia, Biorad) was equilibrated with buffer A (Tris-Cl 50 mM pH 7.4, NaCl 150 mM). (B) Supernatant of lyzed bacteria was introduced through the column. (C) Washing with 5% of buffer B. (D) Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, pressure in brown, temperature in cyan, and concentration of buffer B in green. Flow-rate : 0.5 ml/min. Fractions size : 1 ml.</br></br>

(A) Polystyrene-based Ni-NTA column (Nuvia, Biorad) was equilibrated with buffer A (Tris-Cl 50 mM pH 7.4, NaCl 150 mM). (B) Supernatant of lyzed bacteria was introduced through the column. (C) Washing with 5% of buffer B. (D) Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, pressure in brown, temperature in cyan, and concentration of buffer B in green. Flow-rate : 0.5 ml/min. Fractions size : 1 ml.</br></br>

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We first tested the ability of our protein to bind to cellulose. To do that, we used several types of cellulose: Avicell, Sigmacell, and carboxymethyl-cellulose. As described by Goldstein et al1, we mixed cellulose with an excess of competitor non-specific protein (BSA), and with or without our protein of interest (Fig. 8A). After washing and centrifugation, we harvested proteins from supernatant and the cellulose-based pellet in order to analyze them by SDS-PAGE. We clearly noted that the 25 kDa and 50 kDa proteins were retained by cellulose, instead of the non-specific BSA (Fig. 8B). Indeed, data showed that almost no monomer remained in the supernatant after the second wash, but the pellet contained most of the protein. However, some dimers seem to remain in the second washing supernatant because the initial protein concentration was too high: the binding sites were saturated. The pellet also contains a lot of the dimers. As control, we observed that BSA remained in the supernatant and didn’t bind to cellulose.  Therefore, we can conclude that our protein binds to cellulose.  

We first tested the ability of our protein to bind to cellulose. To do that, we used several types of cellulose: Avicell, Sigmacell, and carboxymethyl-cellulose. As described by Goldstein et al1, we mixed cellulose with an excess of competitor non-specific protein (BSA), and with or without our protein of interest (Fig. 8A). After washing and centrifugation, we harvested proteins from supernatant and the cellulose-based pellet in order to analyze them by SDS-PAGE. We clearly noted that the 25 kDa and 50 kDa proteins were retained by cellulose, instead of the non-specific BSA (Fig. 8B). Indeed, data showed that almost no monomer remained in the supernatant after the second wash, but the pellet contained most of the protein. However, some dimers seem to remain in the second washing supernatant because the initial protein concentration was too high: the binding sites were saturated. The pellet also contains a lot of the dimers. As control, we observed that BSA remained in the supernatant and didn’t bind to cellulose.  Therefore, we can conclude that our protein binds to cellulose.  

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Then, we investigated whether our protein was able to catalyze the biosilification reaction. To do that, we drew inspiration for the 2011 Minnesota iGEM team and their work about Si4 to evaluate the silification process. First, we used a source of silicic acid, the tetraethyl orthosilicate (TEOS), which is an inactive form of silicic acid. By activating it in acidic conditions, we released the free silicic acid (Fig. 9A). After incubation with or without our fusion protein, we determined the quantity of free silicic acid by a spectrophotometric method, since biosilification process consumes silicic acid to form silica (Fig. 9B). We clearly observed a precipitation into the test tube, instead of the negative control (Fig. 10A). By quantifying it by molybdate assay using a standard curve (Fig. 10B), we deduced the corresponding mass of silicic acid left after silification: 33 µg. Before silification, the concentration was 208 µg/ml. The fusion protein led to the production of 175 µg of silica after 2 hours. Therefore, the silification yield after two hours is up to 84% with the protein whereas the yield without the protein is 0% (Fig. 10C). We concluded that our protein worked.   

Then, we investigated whether our protein was able to catalyze the biosilification reaction. To do that, we drew inspiration for the 2011 Minnesota iGEM team and their work about Si4 to evaluate the silification process. First, we used a source of silicic acid, the tetraethyl orthosilicate (TEOS), which is an inactive form of silicic acid. By activating it in acidic conditions, we released the free silicic acid (Fig. 9A). After incubation with or without our fusion protein, we determined the quantity of free silicic acid by a spectrophotometric method, since biosilification process consumes silicic acid to form silica (Fig. 9B). We clearly observed a precipitation into the test tube, instead of the negative control (Fig. 10A). By quantifying it by molybdate assay using a standard curve (Fig. 10B), we deduced the corresponding mass of silicic acid left after silification: 33 µg. Before silification, the concentration was 208 µg/ml. The fusion protein led to the production of 175 µg of silica after 2 hours. Therefore, the silification yield after two hours is up to 84% with the protein whereas the yield without the protein is 0% (Fig. 10C). We concluded that our protein worked.   

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Our patch aims at detecting mosquito-borne pathogen antigens. Since the composite patch-protein was not completely assembled, we characterized the efficiency of our detection method by using commercial membranes of PVDF or nitrocellulose. The detection method we designed was based on the conjugation of antigens with the HRP, and the capture of these conjugated antigens by specific antibodies.  We revealed interactions by incubating membranes with the HRP’s substrate.</br></br>

Our patch aims at detecting mosquito-borne pathogen antigens. Since the composite patch-protein was not completely assembled, we characterized the efficiency of our detection method by using commercial membranes of PVDF or nitrocellulose. The detection method we designed was based on the conjugation of antigens with the HRP, and the capture of these conjugated antigens by specific antibodies.  We revealed interactions by incubating membranes with the HRP’s substrate.</br></br>

First, we tested whether our detection method was working by using single purified viral protein of yellow fever virus (YFVp). By incubating coated membranes with conjugated HRP-BSA, single EZ, or non conjugated YFVp, we didn’t observe any signal (Fig. 11). However, in presence of conjugated HRP-YFVp, a dark spot appeared (Fig. 11). Thus showing that we were able to detect YFVp by this immunodetection technique. </br></br>

First, we tested whether our detection method was working by using single purified viral protein of yellow fever virus (YFVp). By incubating coated membranes with conjugated HRP-BSA, single EZ, or non conjugated YFVp, we didn’t observe any signal (Fig. 11). However, in presence of conjugated HRP-YFVp, a dark spot appeared (Fig. 11). Thus showing that we were able to detect YFVp by this immunodetection technique. </br></br>

Then, in the presence of an excess of non-specific proteins such as BSA, we showed that we maintained the specificity of our detection method (data not shown). </br></br>

Then, in the presence of an excess of non-specific proteins such as BSA, we showed that we maintained the specificity of our detection method (data not shown). </br></br>

To determine whether mosquito proteins can interfere with the specific interaction between viral proteins and specific antibodies, we coated PVDF membranes with CHIKV-specific antibodies, and we conjugated CHIKV envelope protein in presence of an excess of mosquito proteins (from non infected mosquitoes). Then, we incubated coated membranes with conjugated mosquito proteins in the presence or absence of CHIKV envelope protein. We noted a basal level of interaction between the CHIKV-specific antibody 3E4 and mosquito proteins (Fig. 12). However, we noted an important increase of signal when CHIKV envelope protein is present in the mixture. It is obvious that we have to improve the specificity of our detection method in the presence of mosquito lysate. </br></br>

To determine whether mosquito proteins can interfere with the specific interaction between viral proteins and specific antibodies, we coated PVDF membranes with CHIKV-specific antibodies, and we conjugated CHIKV envelope protein in presence of an excess of mosquito proteins (from non infected mosquitoes). Then, we incubated coated membranes with conjugated mosquito proteins in the presence or absence of CHIKV envelope protein. We noted a basal level of interaction between the CHIKV-specific antibody 3E4 and mosquito proteins (Fig. 12). However, we noted an important increase of signal when CHIKV envelope protein is present in the mixture. It is obvious that we have to improve the specificity of our detection method in the presence of mosquito lysate. </br></br>