Difference between revisions of "Team:NCKU Tainan/Team"

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<div class="col-6"><li  class="nav-item" id="exp1"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#poverview">- Overview </a></li></div>
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<div class="col-6"><li  class="nav-item" id="exp1"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#poverview">- Principal Investigator </a></li></div>
 
<div class="col-4"><img id="testtest1" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier" style="left=-1rem"></div>
 
<div class="col-4"><img id="testtest1" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier" style="left=-1rem"></div>
 
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<div class="col-6"><li  class="nav-item" id="exp2"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#pcontact">- Contact lens </a></li></div>
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<div class="col-6"><li  class="nav-item" id="exp2"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#pcontact">- Advisor </a></li></div>
 
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<div class="col-6"><li  class="nav-item" id="exp3"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#pgene">- Gene design </a></li></div>
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<div class="col-6"><li  class="nav-item" id="exp3"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#pgene">- Team Member </a></li></div>
 
<div class="col-4"><img id="testtest3" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier"></div>
 
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<div class="col-6"><li  class="nav-item" id="exp4"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#pdevice">- Device </a></li></div>
 
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<div class="col-4"><img id="testtest4" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier" style="left=-1rem"></div>
 
 
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             </ul>
 
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             <div class="resume-section-content" id="poverview">
 
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                 <h2 class="mb-3">Overview</h2>
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                 <h2 class="mb-3">Principal Investigator</h2>
 
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                        <p>   In our mission to address glaucoma comprehensively, we decided to provide an even more effective treatment for the disease. Inspired by existing treatments using the nitric-oxide (NO) signaling pathway to target the trabecular meshwork and reduce intraocular pressure<sup>[<a href="#ref1" class="linklink">1</a>]</sup>, we came up with a novel treatment based on gaseous nitric oxide. However, since NO has a short half-life of 400 seconds, we are unable to use the gaseous form NO directly in our treatment.</p>
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                        <div class="photo-wrapper d-flex justify-content-center flex-wrap w-100">
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                            <!--I-Son Ng-->
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                            <div class="intro-all">
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                              <img src="https://static.igem.org/mediawiki/2019/thumb/8/8d/T--NCKU_Tainan--Team_ng_1.jpg/398px-T--NCKU_Tainan--Team_ng_1.jpg" title="click" class="normal w-100">
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                              <img src="https://static.igem.org/mediawiki/2019/thumb/a/a5/T--NCKU_Tainan--Team_ng_2.jpg/399px-T--NCKU_Tainan--Team_ng_2.jpg" class="hover d-none w-100">
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                              <div class="intro d-none">
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                                <h3 class="card-title">Dr. I-Son Ng</h3>
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                                <p class="card-text">
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                                  <b>Associate Professor - Department of Chemical Engineering</b><br>
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                                  Dr. Ng is devoted to the study of synthetic biology, genetic and enzymatic engineering, biorefinery and bioremediation. She has developed a robust and efficient platform to explore the novel and functional enzymes by genetic and proteomics approach.<br>
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                                </p>
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                            <!--I-Hsiu Huang-->
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                            <div class="intro-all">
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                              <div class="intro d-none">
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                                <h3 class="card-title">Dr. I-Hsiu Huang</h3>
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                                <p class="card-text">
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                                  <b>Associate Professor - Department of Microbiology and Immunology</b><br>
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                                  Dr. Huang aims to identify novel therapeutic and preventive measures against <i>Clostridium difficile</i>, a leading cause of antibiotic-associated diarrhea worldwide. He is also working on identifying the role of oral microbiome in oral cancer progression and proliferation.<br>
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                                </p>
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                            <!--Masayuki Hashimoto-->
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                            <div class="intro-all">
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                              <img src="https://static.igem.org/mediawiki/2019/thumb/a/a1/T--NCKU_Tainan--Team_masa_2.jpg/398px-T--NCKU_Tainan--Team_masa_2.jpg" title="click" class="hover d-none w-100">
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                              <div class="intro d-none">
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                                <h3 class="card-title">Dr. Masayuki Hashimoto</h3>
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                                <p class="card-text">
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                                  <b>Associate Professor - Institute of Molecular Medicine</b><br>
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                                  I'm a bacterial genetist, majorly study E. coli and B. subtilis. It's my pleasure to join the NCKU iGEM team. I'm really enjoying the project with young students. They are so active, so I'm also stimulated by them. In addition, I sometimes enjoy triathlon race.<br>
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                                </p>
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                            <!--Han-Ching Wang-->
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                            <div class="intro-all">
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                              <img src="https://static.igem.org/mediawiki/2019/thumb/e/e4/T--NCKU_Tainan--Team_hanchin_1.jpg/399px-T--NCKU_Tainan--Team_hanchin_1.jpg" title="click" class="normal w-100">
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                              <img src="https://static.igem.org/mediawiki/2019/thumb/e/ee/T--NCKU_Tainan--Team_hanchin_2.jpg/399px-T--NCKU_Tainan--Team_hanchin_2.jpg" title="click" class="hover d-none w-100">
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                              <div class="intro d-none">
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                                <h3 class="card-title">Dr. Han-Ching Wang</h3>
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                                <p class="card-text">
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                                  <b>Professor - Department of Biotechnology and Bioindustry Sciences</b><br>
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                                  Dr. Wang has a goal of using a science-based approach to manage and optimize aquaculture practices. She believes that this is very important for the eco-friendly sustainable shrimp aquaculture industry.<br>
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                 <h2 class="mb-3">Contact lens</h2>
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                 <h2 class="mb-3">Advisor</h2>
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                        <p>    Literature research stated that the increase in patients’ IOP will result in the deformation of cornea, further leading to a structural change in contact lens<sup>[<a href="#ref2" class="linklink">2</a>]</sup>. We utilized this phenomenon to design a pair of contact lenses that structurally change in response to fluctuations in intraocular pressure.</p>
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                        <p>    Our contact lens will be fitted with a tubular semipermeable chamber that is filled with our engineered bacteria, IPTG, NO precursor - L-arginine, and DAP. The volume change of the chamber will cause water to flow out thus increasing the IPTG concentration inside the chamber. The increase in IPTG will induce the bacteria to produce more Nitric Oxide Synthase, which can then convert L-arginine into nitric oxide to lower the intraocular pressure (IOP). This structural change is then able to induce dynamic drug delivery.</p>
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                <h3 class="mb-0" style="margin-top: 1rem;">IOP simulation experiment</h3>
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                        <p>    To prove the concept of our contact lens and our device, we designed an IOP simulation experiment with porcine eye.By changing the drip bag’s height, water pressure will directly increase IOP in the porcine eye, enabling precise control of IOP for experiments<sup>[<a href="#ref3" class="linklink">3</a>]</sup><sup>[<a href="#ref4" class="linklink">4</a>]</sup>.</p>
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                        <p>    For more details, please visit <a href="https://2020.igem.org/Team:NCKU_Tainan/Proof_Of_Concept" class="linkinthetext">proof of concept</a> and <a href="https://2020.igem.org/Team:NCKU_Tainan/Hardware" class="linkinthetext">hardware.</a></p>
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                <a href="https://static.igem.org/mediawiki/2020/4/4e/T--NCKU_Tainan--design_contact_lens.png" target="_blank" style="width:60%"><img src="https://static.igem.org/mediawiki/2020/4/4e/T--NCKU_Tainan--design_contact_lens.png" alt="" title="" style="width:100%"></a>
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                <figcaption class="caption-design">Fig. 1. The structure and design of contact lens.</figcaption>
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                 <h2 class="mb-3">Gene Design</h2>
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                        <p>    In order for our bacteria to reduce intraocular pressure, we planned to engineer our bacteria to have the ability to produce Nitric Oxide Synthase (NOS)<sup>[<a href="#ref5" class="linklink">5</a>]</sup>, an enzyme that can convert L-arginine into NO.</p>
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                        <p>    For biosafety, we engineered our bacteria to overexpress <i>csgD</i> and <i>csgA</i> for securing bacteria onto the contact lens by increasing binding affinity between bacteria and lens.</p>
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              <h3 class="mb-0" style="margin-top: 1rem;"><i>Nitric Oxide Synthases</i> (<i>NOS</i>)</h3>
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                        <p>    During literature research, we found out that Bacillus subtilis carries <i>Nitric Oxide Synthases</i>(<i>NOS</i>) and has the ability to produce NO, which is responsive to oxidative stress. So we cloned this gene from Bacillus subtilis' genome and designed a new biobrick which we then incorporated into our chassis, WM3064, allowing it to produce NO.</p>
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                        <p>    In order to dynamically express NOS as patients’ IOP fluctuate, we put <i>NOS</i> under the control of <i>T7</i> promoter and a <i>lacO</i> binding site, which can be controlled by IPTG-inducible <i>T7</i> RNA polymerase provided by another plasmid PDT7 (Plasmid Drive <i>T7</i> RNA polymerase). As previously mentioned, the IPTG concentration inside the ring-like compartment will fluctuate according to the patients’ IOP, leading to dynamic expression of <i>NOS</i>.</p>
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                <a href="https://static.igem.org/mediawiki/2020/3/3b/T--NCKU_Tainan--design_NOS.gif" target="_blank" style="width:60%"><img src="https://static.igem.org/mediawiki/2020/3/3b/T--NCKU_Tainan--design_NOS.gif" alt="" title="" style="width:100%"></a>
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                <figcaption class="caption-design">Fig. 2. Plasmid design for <i>NOS</i>.</figcaption>
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                <h4 class="mb-0">Functional Test</h4>
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                        <p>    We tested the kinetics of the enzyme using a NOS assay kit, which utilizes Griess reagents to react with NO and generate colorimetric readouts by measuring O.D.540 value. For the purpose of controlling the production of NOS, we induced bacteria with different concentrations of IPTG and cultured them for different period times.</p>
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                <h3 class="mb-0" style="margin-top: 1rem;">Biosafety</h3>
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                <h4 class="mb-0">DAP-deficient strain</h4>
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                        <p>    We chose <i>E. coli</i> WM3064 as our chassis, which lacks the essential gene <i>dapA</i>. This gene encodes for 4-hydroxy-tetrahydrodipicolinate synthase that is critical to the production of lysine through the DAP pathway<sup>[<a href="#ref6" class="linklink">6</a>]</sup>. Lysine is an essential amino acid in animals, including humans, but can be synthesised de novo in bacteria, lower eukaryotes and plants for utilisation in protein and peptidoglycan cell wall synthesis<sup>[<a href="#ref1" class="linklink">7</a>]</sup>. Without this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) to survive.</p>
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                <a href="https://static.igem.org/mediawiki/2020/3/3c/T--NCKU_Tainan--design_DAP.gif" target="_blank" style="width:60%"><img src="https://static.igem.org/mediawiki/2020/3/3c/T--NCKU_Tainan--design_DAP.gif" alt="" title="" style="width:100%"></a>
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                <figcaption class="caption-design">Fig. 3. Design of biosafety chassis.</figcaption>
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                </figure>
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                <h4 class="mb-0">Functional Test</h4>
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                        <p>    To test whether the bacteria will survive without exogenous DAP, we made plates with and without DAP. After streaking our engineered bacteria onto these plates, we can demonstrate the result by checking its phenotype. Furthermore, we ran a SDS-PAGE to confirm the function of the <i>T7</i> expression system in our engineered WM3064.</p>
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                    </div>
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                <h4 class="mb-0">Overexpression <i>csgD</i> and <i>csgA</i></h4>
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                <div class="d-flex flex-column flex-md-row justify-content-between mb-2">
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                        <p>    Bacteria biofilm has been shown to exhibit extraordinary ability to help bacteria bind to biotic and abiotic surfaces[measurement reference]. We exploited this property to design one of the biosafety measures.</p>
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                      <p>    We engineered our bacteria to overexpress <i>CsgD</i>, a master transcription regulator of biofilm formation, and <i>CsgA</i>, the major subunit of curli fibers. Overexpression of these two proteins have been reported to increase biofilm formation[measurement reference], which we anticipated to help the bacteria bind to the contact lenses more securely, thus preventing the leakage of bacteria if the contact lens encounters any damage.</p>
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                <h4 class="mb-0">Functional Test</h4>
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                        <p>    We first characterized the biofilm formation using conventional congo red staining, then we developed a simple method to assess the binding ability of our engineered bacteria. For more information, please visit our <a href="https://2020.igem.org/Team:NCKU_Tainan/Measurement" class="linkinthetext">measurement page</a>.</p>
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                <h3 class="mb-0" style="margin-top: 1rem;">Growth switch</h3>
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                <div class="d-flex flex-column flex-md-row justify-content-between mb-2">
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                        <p>    There are several things that need to be considered before selling Eye kNOw as a product. Since Eye kNOw won’t be used by the patient immediately after being manufactured, we designed a growth switch in order to control bacteria growth in different stages of our product lifetime. We were inspired by the work of <a href="https://2019.igem.org/Team:NUS_Singapore" class="linkinthetext">iGEM NUS 2019</a> who used a toxin-antitoxin system, <i>hicA</i>-<i>hicB</i>, to control the growth of bacteria. By manipulating the toxin-antitoxin ratio in a bacteria, we can determine when the bacteria should hibernate or grow.</p>
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                        <p>    In our design, the <i>hicB</i> antitoxin is constitutively expressed at a basal level, while the <i>hicA</i> toxin is controlled by arabinose inducible promoter. The entire <i>hicA</i> cassette is flanked with <i>FRT</i> sites, which can later be deleted by the FLP recombinase. We also added a heat-activated <i>FRT</i>-FLP recombinase system from pCP20 as an inducible switch. This design enables us to control the bacteria growth in three stages - production, storage and medication.</p>
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                <div class="d-flex flex-column flex-md-row justify-content-between mb-2">
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                        <p>    The production stage is when we are culturing our bacteria, so we need the bacteria to be able to grow normally. After the production process, the bacteria needs to be stored in the contact lens until it can be used. During the storage stage, <i>hicA</i> will be induced to hibernate the bacteria. The last stage is the medication stage, during which the contact lens will be used. When the contact lens is placed on the patient’s eye, the body temperature will activate the recombinase system and delete the hicA cassette, which will cause the bacteria to resuscitate and start producing the therapeutic agent.</p>
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                <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3">
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                <a href="https://static.igem.org/mediawiki/2020/9/9a/T--NCKU_Tainan--design_HicAB.gif" target="_blank" style="width:60%"><img src="https://static.igem.org/mediawiki/2020/9/9a/T--NCKU_Tainan--design_HicAB.gif" alt="" title="" style="width:100%"></a>
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                <figcaption class="caption-design">Fig. 4. Plasmid design for grow switch</figcaption>
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                <h4 class="mb-0">Functional Test</h4>
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                        <p>    To verify the function of the FLP-<i>FRT</i> system, we will culture the bacteria with three plasmids, containing <i>hicA</i>, <i>hicB</i>, and <i>CI857</i> and <i>FLP</i> genes respectively. As the temperature rises to 42<sup>o</sup>C, <i>CI857</i> gene will be degraded, activating the <i>FLP</i> gene and deleting the <i>hicA</i> gene. If the <i>hicA</i> gene is present, the O.D.600 value will not increase since HicA protein represses the growth of bacteria. Hence, we can verify the function of the growth switch by measuring O.D. 600 value.</p>
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        <hr class="hrmar" />     
 
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        <section class="resume-section">
 
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            <div class="resume-section-content" id="pdevice">
 
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                <h2 class="mb-3">Device</h2>
 
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                <h3 class="mb-0" style="margin-top: 1rem;">IOP Detector</h3>
 
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                <div class="d-flex flex-column flex-md-row justify-content-between mb-2">
 
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                        <p>    Since there are no early symptoms of glaucoma, the public is left unaware of its presence. Therefore, early detection is needed, so we developed a brand-new IOP detector - Eye Screen. By transmitting ultrasonic waves to the patient’s cornea and analyzing the reflected signal, we can get IOP readings immediately. With Eye Screen, we can quickly find people at high risk of glaucoma, without direct contact with the eyes.</p>
 
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                    </div>
 
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                </div>
 
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                <h4 class="mt-1">Functional Test</h4>
 
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                    <div class="flex-grow-1">                     
 
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                        <p>    To validate the function of Eye Screen, we adopted a gravity model to control the IOP of porcine eyeballs using the trocar system via microincision vitrectomy surgery. By adjusting the height of the saline bag connected to the eyeball, we can measure the IOP by calculating the difference in height of the saline and the eyeball. We then tested whether the amplitude of reflected signals can be proportional to the IOP according to the change in IOP.</p>
 
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        <hr class="hrmar" />
 
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        <section class="resume-section" >
 
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          <div class="resume-section-content" id="ref">
 
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            <h2>References</h2>
 
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              <ol>
 
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                <li id="ref1">Muenster S, Lieb WS, Fabry G, et al. The Ability of Nitric Oxide to Lower Intraocular Pressure Is Dependent on Guanylyl Cyclase. <i>Investigative Opthalmology & Visual Science.</i> 2017;58(11):4826.</li>
 
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                <li id="ref2">Lam A. The effect of an artificially elevated intraocular pressure on the central corneal curvature. <i>Ophthalmic and Physiological Optics.</i> 1997;17(1):18-24.</li>
 
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                <li id="ref3">Chen G-Z, Chan I-S, Leung LKK, Lam DCC. Soft wearable contact lens sensor for continuous intraocular pressure monitoring. <i>Medical Engineering & Physics.</i> 2014;36(9):1134-1139.</li>
 
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                <li id="ref4">Zhang J, Zhang Y, Li Y, et al. Correlation of IOP with Corneal Acoustic Impedance in Porcine Eye Model. <i>BioMed Research International.</i> 2017;2017:1-6.</li>
 
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                <li id="ref5">BRENDA - Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH). Brenda-enzymes.org. <a href="https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM." target="_blank" class="linklink">https://www.brenda-enzymes.org/enzyme.php?ecno=1.14.13.39#pH%20OPTIMUM.</a> Published 2020. Accessed September 9, 2020.</li>
 
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                <li id="ref6">Dante RA, Neto GC, Leite A, Yunes JA, Arruda P. Plant <i>Molecular Biology.</i> 1999;41(4):551-561.</li>
 
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                <li id="ref7">McLennan N, Masters M. GroE is vital for cell-wall synthesis. <i>Nature.</i> 1998;392(6672):139-139.</li>
 
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Revision as of 08:49, 25 October 2020


Team

All of our cute and hardworking members

Principal Investigator

Dr. I-Son Ng

Associate Professor - Department of Chemical Engineering
Dr. Ng is devoted to the study of synthetic biology, genetic and enzymatic engineering, biorefinery and bioremediation. She has developed a robust and efficient platform to explore the novel and functional enzymes by genetic and proteomics approach.

Dr. I-Hsiu Huang

Associate Professor - Department of Microbiology and Immunology
Dr. Huang aims to identify novel therapeutic and preventive measures against Clostridium difficile, a leading cause of antibiotic-associated diarrhea worldwide. He is also working on identifying the role of oral microbiome in oral cancer progression and proliferation.

Dr. Masayuki Hashimoto

Associate Professor - Institute of Molecular Medicine
I'm a bacterial genetist, majorly study E. coli and B. subtilis. It's my pleasure to join the NCKU iGEM team. I'm really enjoying the project with young students. They are so active, so I'm also stimulated by them. In addition, I sometimes enjoy triathlon race.

Dr. Han-Ching Wang

Professor - Department of Biotechnology and Bioindustry Sciences
Dr. Wang has a goal of using a science-based approach to manage and optimize aquaculture practices. She believes that this is very important for the eco-friendly sustainable shrimp aquaculture industry.

Advisor

Team Member

e-mail : igem.club@gs.ncku.edu.tw
phone : 06-2757575