<h5id="1.2.1"class="anchor">Cycle 1 - Expressing transcription factors for mercury, lead, cadmium, and arsenic in E. coli TOP10 </h5>
<h5id="1.2.1"class="anchor">Cycle 1 - Expressing transcription factors for mercury, lead, cadmium, and arsenic in <i>E. coli</i> TOP10 </h5>
<p><b>Design:</b></p>
<p>ArsR, PbrR, MerR and mut_MerR are transcription factors (TFs) that can bind DNA and arsenic, lead, mercury or cadmium respectively. When no metal is present, the TFs can bind to specific DNA sites, preventing any enzymes from moving along the sequence. However, when bound to a metal the TFs unbind the DNA. As such, transcription can be controlled depending on the presence of heavy metals in the reaction mixture. </p>
<p><b>Build:</b></p>
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<p>The sfGFP fluorescence intensities of the initial lysates and supernatants after incubation of the hydrogel and silica beads were measured using a microplate reader. The change in fluorescence intensity indicates the proportion of recombinant sfGFP immobilised to the solid platforms pelleted and separated from the supernatant. For verifying the test, the experiment was replicated using the control lysate containing sfGFP without CBD fusion.</p>
<p><b>Learn:</b></p>
<p>There was no reduction in fluorescence intensity for the SB7-sfGFP control samples, suggesting that the SB7 peptide has little to no affinity for silica beads.</p>
<p>The fluorescence intensities for both CBD-sfGFP and the non-tagged sfGFP control samples dropped drastically, indicating that the CMC-CA hydrogels capture proteins non-selectively which further reinforces the results of our protein absorption test from our previous engineering cycle. The initial fluorescence intensity of the lysate containing CBD-sfGFP was significantly lower than that of the lysate containing the non-tagged sfGFP control, indicating that CBD-fusion may have a suppressive effect on either sfGFP expression in E. coli or its fluorescence. </p>
<p>The fluorescence intensities for both CBD-sfGFP and the non-tagged sfGFP control samples dropped drastically, indicating that the CMC-CA hydrogels capture proteins non-selectively which further reinforces the results of our protein absorption test from our previous engineering cycle. The initial fluorescence intensity of the lysate containing CBD-sfGFP was significantly lower than that of the lysate containing the non-tagged sfGFP control, indicating that CBD-fusion may have a suppressive effect on either sfGFP expression in <i>E. coli</i> or its fluorescence. </p>
<p>Therefore, our CMC-CA hydrogels combined with CBD-tagged proteins (3C hydrogels) are a much more effective method for immobilising proteins compared to displaying silica-affinity-tagged proteins on silica beads. </p>
<p>However, although our hydrogels significantly trapped the CBD-sfGFP, this test still does not clearly show whether the cellulose hydrogel matrix has a significant selective affinity for the CBD compared to other proteins.</p>
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<p><b>Learn:</b></p>
<ul>
<li>Unexpectedly, the negative control hydrogels captured a slightly higher amount of Zn(II) (i.e. slightly larger Zn(II) concentration reduction in solution) than our metallothionein-displaying 3C hydrogels. A possible explanation for this is incubating absorbent hydrogels in lysates may have lead to non-selective immobilisation of many proteins, and there are many Zn(II)-binding proteins in cells other than metallothioneins. Another explanation is that the washing step may not be adequate for removing the lysis buffer components that include metal-chelating compounds EDTA and DTT.</li>
<li>Our metallothionein-displaying 3C hydrogels captured a slightly higher amount of Ni(II) (i.e. slightly larger Ni(II) concentration reduction in solution) than the negative control hydrogels. We speculate that the Ni(II) binding capacity of our hydrogels after decoration with CBD-MT was improved by only a small margin due to low expression of CBD-MT in E. coli (thus, may be present at relatively low amounts in the lysates) which is a factor we were not able to measure due to time constraints.</li>
<li>Our metallothionein-displaying 3C hydrogels captured a slightly higher amount of Ni(II) (i.e. slightly larger Ni(II) concentration reduction in solution) than the negative control hydrogels. We speculate that the Ni(II) binding capacity of our hydrogels after decoration with CBD-MT was improved by only a small margin due to low expression of CBD-MT in <i>E. coli</i> (thus, may be present at relatively low amounts in the lysates) which is a factor we were not able to measure due to time constraints.</li>
</ul>
<p>Overall, our engineered 3C hydrogels have the potential to capture heavy metal ions to at least a higher extent compared to typical biopolymer-based hydrogels without immobilised metallothioneins, but further measures (especially using purified CBD-MT instead of its host E. coli lysate) could be taken to improve them.</p>
<p>Overall, our engineered 3C hydrogels have the potential to capture heavy metal ions to at least a higher extent compared to typical biopolymer-based hydrogels without immobilised metallothioneins, but further measures (especially using purified CBD-MT instead of its host <i>E. coli</i> lysate) could be taken to improve them.</p>
