Since the cellulose-based patch alone could not detect mosquito-borne pathogens, we used a <B>multifunctional fusion protein</B> to make it stronger. This protein is bonded to the cellulose-based patch and made it stronger by <B>bio-condensing silicic acid</B> into silica and fixing <B>specific antibodies</B>. Therefore, we took advantage of the silica-binding peptide (designated by Si4), the B domain of staphylococcal protein A (designated by BpA), and the cellulose-binding domain of cellulose-binding protein A (designated by CBPa). Since it was important that Si4 and BpA were not close in order to avoid steric hindrance of the paratope by condensated silica, we designed the fusion protein as Si4-CBPa-BpA (Fig 2).</br></br></br></br>

To produce the protein, we had to assemble the corresponding genes of each part of the fusion protein by using preexisting iGEM BioBricks. In fact, we assembled <a href="http://parts.igem.org/Part:BBa_K1028000"><B>BBa_K1028000</B></a> (encoding for Si4, from <a href="https://2013.igem.org/Team:Leeds"><B>iGEM Leeds 2013</B></a>), <a href="http://parts.igem.org/Part:BBa_K863110"><B>BBa_KBBa_K863110</a> (encoding for CBPa, from <a href="https://2012.igem.org/Team:Bielefeld-Germany"><B>iGEM Bielefeld-Germany 2012)</B></a>, and <a href="http://parts.igem.org/Part:BBa_K103003"><B>BBa_Khref=BBa_K103003</a>(encoding for BpA, from <a href="https://2008.igem.org/Team:Warsaw"><B>iGEM Warsaw 2008</B>)</a>, and separated them by using <B>flexible linkers</B> in order to preserve their conformation and their biological activity. To make our protein easy to purify, the sequence of a HisTag was added at the 5’ end, followed by the TEV protease-specific cleavage site sequence to allow the removal of the His-Tag after purification. To be certain of our protein’s expression, we intentionally added ATG initiation codons at the beginning and TAA stop codons at the end of the sequence. Next, cis-regulating elements, such as T7 terminator and T7 promoter, were taken from the pET43.1a(+) vector to flank the composite sequence, and iGEM prefix and suffix were added. BamH I and Hind III restrictions sites were added to the beginning and the end of final sequence, respectively2 (Fig 3). </br></br></br></br>

We optimized the coding sequence for expression in <i>Escherichia coli</i> with Geneious software, and we took the precaution of avoiding restriction sites of iGEM prefix and suffix. By taking advantage of the free 20bp offered by Integrated DNA Technologies (IDT), we sent several sequences to be synthesized which encoded the fusion protein and several control sequences (Table 1).  </br></br>

Once we received DNA sequences, we amplified them by polymerase chain reaction (PCR) and cloned them into TOPO vectors[3] (Fig 4). Since Taq polymerase contains a terminal-desoxynucleotidyl transferase activity, adenine were added on each extremity of the PCR products, allowing TOPO cloning. Resulting “TOPO-insert” plasmids were introduced into competent TOP 10 <i>Escherichia coli</i> by heat-shock transformation, and bacteria were grown onto LB plates added of ampicillin to increase plasmid amount. Since resistance gene to ampicillin is present in the TOPO vector, only transformed bacteria were selected. After growth, bacteria were harvested and plasmidic DNA extracted. Following restriction enzymatic digestion by using XbaI/HindIII enzymes couple and electrophoresis, we checked that extracted plasmids contain insert, and extracted it from the gel. After that, we cloned this insert into digested and dephosphorylated expression vector pET43.1a(+), and repeat the same procedure until the plasmidic DNA extraction and verification by electrophoresis. </br></br>

To produce our protein, we transformed specifically dedicated BL21(DE3) <i>Escherichia coli</i> with “pET43.1a(+)insert” to produce high amounts of proteins. We cultured them and when OD600nm was close to 0.7, we induced our protein expression by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) in the growth medium[4]. It was important to be sure that our protein expression was not constitutive since we didn’t know the potential toxic effect of it onto the bacteria metabolism. Bacteria were harvested and our own protein purified by Fast Pressure Liquid Chromatography (FPLC) by using a Nickel-tagged and plastic-based column[5]. We detected the presence of our protein by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration into fractions of interest was determined by Bradford assay. In order to be sure that it is our own fusion protein, we sent SDS-PAGE bands to mass spectrometry platform of Institut Pasteur, and performed Western Blot by using anti-HisTag antibody (Fig. 4). Moreover, Si4 and CBPa parts were independently validated and characterized into our fusion protein by performing functional experiments: biosilification and cellulose-binding tests. Unfortunately, we didn’t have the time to test BpA part.</br></br></br></br>