<p>The primary challenge with microbial opsins in the past was their limited sensitivity to light. This meant that a significant number of photons had to interact with these proteins to trigger a conformational change. Only after this change could the ion channel within the opsin protein open, subsequently initiating a neural response. Consequently, using these proteins in genetic therapy required patients to wear specialized goggles [1]. These goggles amplified the light intensity of the surrounding environment, compensating for the low sensitivity of the opsins.</p>
<p>The primary challenge with microbial opsins in the past was their limited sensitivity to light. This meant that a significant number of photons had to interact with these proteins to trigger a conformational change. Only after this change could the ion channel within the opsin protein open, subsequently initiating a neural response. Consequently, using these proteins in genetic therapy required patients to wear specialized goggles <b>[1]</b>. These goggles amplified the light intensity of the surrounding environment, compensating for the low sensitivity of the opsins.</p>
<p>Our project's primary aim was to evolve these opsins. We wanted to create genetic variants that responded to light with enhanced sensitivity. In simpler terms, these evolved opsins would open their ion channels with far fewer photons. The end goal was to harness these proteins for genetic therapy, eliminating the need for patients to rely on light-boosting goggles. Another facet of our project was to shift the light absorption of these opsins. By evolving them to absorb light closer to the red end of the visible spectrum, instead of the blue, we intended to reduce potential cellular damage. This shift was crucial because blue light carries more energy and can be harmful to cells over prolonged exposure.</p>
<p>Given the optogenetic foundation of our project, a specific stage in the evolution process required us to shine light on the liquid cultures. This was to prompt the opsins to open their sodium (Na<sup>+</sup>) channels. We then detected this influx of sodium using a fluorescent dye. Once we successfully detected an opsin channel opening, our subsequent tests involvs illuminating our cultures with decreasing light intensities post-evolution. This step is critical in pinpointing which genetic variant underwent a mutation during random mutagenesis, granting it enhanced light sensitivity. In the later stages, we also aim at identifing variants that had evolved to absorb light of a shifted wavelength.</p>
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<h4id="ewod-intro"style="text-align: center; margin-top: 60px;">Moving a Droplet Using EWOD</h4>
<hr>
<p>By applying voltage to an adjacent electrode while turning off the one beneath the droplet, the droplet starts stretching towards the activated electrode. Due to surface tension, it's eventually pulled in that direction entirely [2]. Harnessing this principle, we could control droplet movement, mix, and even split them with a smart electrode layout. Plus, we could add different components to the platform to create specialised functional zones, such as heating zones for PCR or cell incubation liquid culture, cold storage, magnetic zones for functionalized magnetic beads-based molecular purification, optical analysis for droplet content quantification comparable to a nanodrop apparatus, etc [3].</p>
<p>By applying voltage to an adjacent electrode while turning off the one beneath the droplet, the droplet starts stretching towards the activated electrode. Due to surface tension, it's eventually pulled in that direction entirely <b>[2]</b>. Harnessing this principle, we could control droplet movement, mix, and even split them with a smart electrode layout. Plus, we could add different components to the platform to create specialised functional zones, such as heating zones for PCR or cell incubation liquid culture, cold storage, magnetic zones for functionalized magnetic beads-based molecular purification, optical analysis for droplet content quantification comparable to a nanodrop apparatus, etc <b>[3]</b>.</p>
<h4id="project-vision"style="text-align: center;">Our Vision for the Platform</h4>
<p>In our project, we aimed to demonstrate that the envisioned platform could go beyond merely aiding the automation of directed evolution protocols. We believed that it held vast potential as an ultra-high-throughput, multifunctional lab automation system. With the rise of AI, we opted to make our hardware compatible with machine learning algorithms. Our goal was to highlight the potential future of AI-driven research platforms.</p>
<h4id="platform-design"style="text-align: center;">Platform Design and Capabilities</h4>
<p>When it came to the platform's design and capabilities, we began by constructing a basic version (<b>Figure 9</b>). However, we didn't compromise on performance. While we utilized straightforward Mosfet transistor controls to power individual electrodes [4], our choice of polymers set our work apart. This combination enabled us to showcase a pioneering electrowetting platform in the iGEM competition. Looking at past projects, there has never been a digital microfluidic apparatus demonstration capable of moving droplets at such high speed with no missteps.</p>
<p>When it came to the platform's design and capabilities, we began by constructing a basic version (<b>Figure 9</b>). However, we didn't compromise on performance. While we utilized straightforward Mosfet transistor controls to power individual electrodes <b>[4]</b>, our choice of polymers set our work apart. This combination enabled us to showcase a pioneering electrowetting platform in the iGEM competition. Looking at past projects, there has never been a digital microfluidic apparatus demonstration capable of moving droplets at such high speed with no missteps.</p>
<p>A crucial component of our design was the dielectric layer, made of a 12um ETFE film [5], a teflon analog. The thickness of this layer is critical for effective droplet movement; thinner layers typically yield better results [6].</p>
<p>A crucial component of our design was the dielectric layer, made of a 12um ETFE film <b>[5]</b>, a teflon analog. The thickness of this layer is critical for effective droplet movement; thinner layers typically yield better results <b>[6]</b>.</p>
<p>The speed of the liquid droplet is influenced by:</p>
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<imgsrc="https://static.igem.wiki/teams/4601/wiki/page-hardware/sans-titre-2.png"alt="Fusion 360 3D printed enclosure design"class="img-fluid mx-auto mt-4"width="300px"style="border-radius: 20px;">
</div>
<p>In which θ signifies the contact angle when subjected to external electric potential V, θ<sub>e</sub> represents the stable contact angle when V equals 0 V, ε<sub>0</sub> stands for the vacuum's permittivity, ε<sub>d</sub> is the dielectric layer's permittivity, γ defines the boundary tension between the droplet and the adjacent insulating liquid, and d refers to the dielectric layer's thickness [7].</p>
<p>In which θ signifies the contact angle when subjected to external electric potential V, θ<sub>e</sub> represents the stable contact angle when V equals 0 V, ε<sub>0</sub> stands for the vacuum's permittivity, ε<sub>d</sub> is the dielectric layer's permittivity, γ defines the boundary tension between the droplet and the adjacent insulating liquid, and d refers to the dielectric layer's thickness <b>[7]</b>.</p>
<p>As our goal was to demonstrate the potential of such technology, we aimed at developing an apparatus capable of multiple hundreds of simultaneous droplets displacements, potentially even thousands and beyond [8].</p>
<p>As our goal was to demonstrate the potential of such technology, we aimed at developing an apparatus capable of multiple hundreds of simultaneous droplets displacements, potentially even thousands and beyond <b>[8]</b>.</p>
<p>To that end, we required the need for a specialized piece of electronic components called a shift register, of the digital to parallel converter type; its goal is to take a digital signal as an input, and connected to high voltage, distribute that power to its many different parallel outputs [9].</p>
<p>To that end, we required the need for a specialized piece of electronic components called a shift register, of the digital to parallel converter type; its goal is to take a digital signal as an input, and connected to high voltage, distribute that power to its many different parallel outputs <b>[9]</b>.</p>
<p>After many testings, we decided to use the HV507 IC [10] for that task, here is how it works (<b>Figures 10 and 11</b>).</p>
<p>After many testings, we decided to use the HV507 IC <b>[10]</b> for that task, here is how it works (<b>Figures 10 and 11</b>).</p>
<li>[1] Häusser M. Optogenetics - The might of light. The New England Journal of Medicine (2021) 385: 1623–1626.</li>
<li>[2] Abdelgawad M. Digital microfluidics: automating microscale liquid handling. IEEE Nanotechnology Magazine (2020) 14: 6–23.</li>
<li>[3] Gach PC, Iwai K, Kim PW, Hillson NJ, Singh AK. Droplet microfluidics for synthetic biology. Lab on a Chip (2017) 17: 3388–3400.</li>
<li>[4] Kothamachu VB, Zaini S, Muffatto F. Role of digital microfluidics in enabling access to laboratory automation and making biology programmable. SLAS Technology (2020) 25: 411–426.</li>
<li>[5] Li J, Kim C-J “CJ.” Current commercialization status of electrowetting-on-dielectric (EWOD) digital microfluidics. Lab on a Chip (2020) 20: 1705–1712.</li>
<li>[6] Nemr CR, Sklavounos AA, Wheeler AR, Kelley SO. Digital microfluidics as an emerging tool for bacterial protocols. SLAS Technology (2023) 28: 2–15.</li>
<li>[7] Samiei E, Tabrizian M, Hoorfar M. A review of digital microfluidics as portable platforms for lab-on a-chip applications. Lab on a Chip (2016) 16: 2376–2396.</li>
<li>[8] Xu X, Cai L, Liang S, Zhang Q, Lin S, Li M, Yang Q, Li C, Han Z, Yang C. Digital microfluidics for biological analysis and applications. Lab on a Chip (2023) 23: 1169–1191.</li>
<li>[9] Yang Y-T, Ho T-Y. Conquering the tyranny of number with digital microfluidics. Frontiers in Chemistry (2021) 9: 676365.</li>
<li>[10] Zhang Y, Liu Y. Advances in integrated digital microfluidic platforms for point-of-care diagnosis: a review. Sensors & Diagnostics (2022) 1: 648–672.</li>
<li><b>[1]</b> Häusser M. Optogenetics - The might of light. The New England Journal of Medicine (2021) 385: 1623–1626.</li>
<li><b>[2]</b> Abdelgawad M. Digital microfluidics: automating microscale liquid handling. IEEE Nanotechnology Magazine (2020) 14: 6–23.</li>
<li><b>[3]</b> Gach PC, Iwai K, Kim PW, Hillson NJ, Singh AK. Droplet microfluidics for synthetic biology. Lab on a Chip (2017) 17: 3388–3400.</li>
<li><b>[4]</b> Kothamachu VB, Zaini S, Muffatto F. Role of digital microfluidics in enabling access to laboratory automation and making biology programmable. SLAS Technology (2020) 25: 411–426.</li>
<li><b>[5]</b> Li J, Kim C-J “CJ.” Current commercialization status of electrowetting-on-dielectric (EWOD) digital microfluidics. Lab on a Chip (2020) 20: 1705–1712.</li>
<li><b>[6]</b> Nemr CR, Sklavounos AA, Wheeler AR, Kelley SO. Digital microfluidics as an emerging tool for bacterial protocols. SLAS Technology (2023) 28: 2–15.</li>
<li><b>[7]</b> Samiei E, Tabrizian M, Hoorfar M. A review of digital microfluidics as portable platforms for lab-on a-chip applications. Lab on a Chip (2016) 16: 2376–2396.</li>
<li><b>[8]</b> Xu X, Cai L, Liang S, Zhang Q, Lin S, Li M, Yang Q, Li C, Han Z, Yang C. Digital microfluidics for biological analysis and applications. Lab on a Chip (2023) 23: 1169–1191.</li>
<li><b>[9]</b> Yang Y-T, Ho T-Y. Conquering the tyranny of number with digital microfluidics. Frontiers in Chemistry (2021) 9: 676365.</li>
<li><b>[10]</b> Zhang Y, Liu Y. Advances in integrated digital microfluidic platforms for point-of-care diagnosis: a review. Sensors & Diagnostics (2022) 1: 648–672.</li>
