Jason Tsai (Talk | contribs) |
Virginiacl07 (Talk | contribs) |
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+ | opacity: 0.2; | ||
+ | width: 100vw; | ||
+ | position: absolute; | ||
+ | left: -2rem; | ||
+ | height: 100vh; | ||
+ | } | ||
+ | section.resume-section-coverphoto { | ||
+ | min-height:93vh; | ||
+ | } | ||
+ | |||
+ | |||
+ | /*needed*/ | ||
section.resume-section { | section.resume-section { | ||
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section.resume-section { | section.resume-section { | ||
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+ | @media only screen and (max-width: 995px) { | ||
+ | #sideNav { | ||
+ | display:none; | ||
+ | |||
+ | } | ||
+ | section.resume-section-coverphoto { | ||
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} | } | ||
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+ | padding-bottom: 1rem; | ||
+ | display: flex; | ||
+ | align-items: center; | ||
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+ | } | ||
+ | .hrmar{ | ||
+ | margin: 0 2rem 0 0; | ||
+ | } | ||
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+ | } | ||
+ | @media only screen and (max-width: 650px) { | ||
+ | .headtitle { | ||
+ | font-size: 2.5rem !important; | ||
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<div class="row navrow"> | <div class="row navrow"> | ||
<div class="col-2"></div> | <div class="col-2"></div> | ||
− | <div class="col-6"><li class="nav-item" id="exp1"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#overview">- Overview </a></li></div> | + | <div class="col-6" style="z-index:2"><li class="nav-item" id="exp1"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#overview">- Overview </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=" | + | <div class="col-4" style="z-index:1"><img id="testtest1" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier" style=""></div> |
</div> | </div> | ||
<div class="row navrow"> | <div class="row navrow"> | ||
<div class="col-2"></div> | <div class="col-2"></div> | ||
− | <div class="col-6"><li class="nav-item" id="exp2"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#results">- Results </a></li></div> | + | <div class="col-6" style="z-index:2"><li class="nav-item" id="exp2"><a class="nav-link js-scroll-trigger nav-list" id="nav-item-list" href="#results">- Results </a></li></div> |
− | <div class="col-4"><img id="testtest2" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier"></div> | + | <div class="col-4" style="z-index:1"><img id="testtest2" src="https://static.igem.org/mediawiki/2020/b/bb/T--NCKU_Tainan--logo_maginifier.png" alt="magnifier"></div> |
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<div class="container-fluid p-0"> | <div class="container-fluid p-0"> | ||
<!-- Description--> | <!-- Description--> | ||
− | <section class="resume-section"> | + | <section class="resume-section resume-section-coverphoto"> |
+ | <img src="https://static.igem.org/mediawiki/parts/9/9e/T--NCKU_Tainan--BBa_K3490011_impruhggjku7y.jpeg" class="coverphoto"> | ||
<div class="resume-section-content" id="pdescription"> | <div class="resume-section-content" id="pdescription"> | ||
<div id="h1-title"> | <div id="h1-title"> | ||
− | <h1 class="mb-0 pl-2" > | + | <h1 class="mb-0 pl-2 headtitle" > |
Improvement | Improvement | ||
<!--<span class="text-primary">Taylor</span>--> | <!--<span class="text-primary">Taylor</span>--> | ||
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− | |||
<!-- Project Background--> | <!-- Project Background--> | ||
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<div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | <div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | ||
<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
− | <p> Therefore, we are trying to improve <a href="http://parts.igem.org/Part:BBa_K805015" target="_blank" class="linkinthetext" | + | <p> Therefore, we are trying to improve <a href="http://parts.igem.org/Part:BBa_K805015" target="_blank" class="linkinthetext">BioBrick BBa_K805015</a> from the <a href="https://2017.igem.org/Team:TAS_Taipei" target="_blank" class="linkinthetext">2017 iGEM TAS-Taipei Team</a><sup>[<a href="#ref2" class="linklink">2</a>]</sup> to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lens despite any damages.</p> |
</div> | </div> | ||
</div> | </div> | ||
<div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | <div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | ||
<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
− | <p> We hypothesized that by adding <i>csgA</i> and <i>pLac</i> to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm | + | <p> We hypothesized that by adding <i>csgA</i> and <i>pLac</i> to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm enables the bacteria to bind securely to the contact lens. Therefore, we add <i>csgA</i> to the biobrick design and change the promoter into <i>pLac</i> as an improvement.</p> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="col-lg "> | <div class="col-lg "> | ||
<figure class="d-flex flex-column justify-content-center align-items-center px-lg-3"> | <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3"> | ||
− | <a href="https://static.igem.org/mediawiki/2020/ | + | <a href="https://static.igem.org/mediawiki/2020/1/1b/T--NCKU_Tainan--Improvment_CsgDA.gif" target="_blank" style="width:65%"><img src="https://static.igem.org/mediawiki/2020/1/1b/T--NCKU_Tainan--Improvment_CsgDA.gif" alt="" title="" style="width:100%"></a> |
<figcaption class="caption-design">Fig. 1. A schematic of our biobrick construction.</figcaption> | <figcaption class="caption-design">Fig. 1. A schematic of our biobrick construction.</figcaption> | ||
</figure> | </figure> | ||
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<div class="resume-section-content" id="results"> | <div class="resume-section-content" id="results"> | ||
<h2 class="mb-3">Experiment Results</h2> | <h2 class="mb-3">Experiment Results</h2> | ||
+ | <h4 class="mb-0" style="margin-top: 1rem;">Congo Red Assay Experiment</h4> | ||
<div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | <div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | ||
<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
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<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
− | <p> Next, to prove that our amount of biofilm production | + | <p> Next, to prove that our amount of biofilm production increased, we did a test by using congo red<sup>[<a href="#ref3" class="linklink">3</a>]</sup> dye to observe the curli expression. We compared the absorbance value of <a href="http://parts.igem.org/Part:BBa_K805015" target="_blank" class="linkinthetext">BBa_K805015</a> and <a href="http://parts.igem.org/Part:BBa_K3490001" target="_blank" class="linkinthetext">BBa_K3490001</a> to see whether the amount of biofilm production increased or not. If the biofilm amount increases, the color of the solution will appear to be darker. So, after overnight culture, we add congo red dye to all the samples. Then we centrifuge to separate the supernatant and precipitate (pellet). By using a microplate reader, we can measure the absorbance value at 500 nm (congo red) and normalized by 600 nm wavelength which represents the amount of bacteria.</p> |
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | < | + | <h4 class="mb-0" style="margin-top: 1rem;">Biofilm Assay Experiment</h4> |
<div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | <div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | ||
<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
− | <p> However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we | + | <p> However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we conducted another experiment to compare the binding affinity of bacteria among control, <i>csgD</i>, and <i>csgA</i>-<i>csgD</i>. We presumed that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we dropped a book from different heights and measuring its OD<sub>600</sub> value. By doing so, we can determine the concentration that represents the binding affinity.</p> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="col-lg "> | <div class="col-lg "> | ||
<figure class="d-flex flex-column justify-content-center align-items-center px-lg-3"> | <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3"> | ||
− | <a href="https://static.igem.org/mediawiki/2020/ | + | <a href="https://static.igem.org/mediawiki/2020/d/d3/T--NCKU_Tainan--measure-pic8.png" target="_blank" style="width:80%"><img src="https://static.igem.org/mediawiki/2020/d/d3/T--NCKU_Tainan--measure-pic8.png" alt="" title="" style="width:100%"></a> |
− | <figcaption class="caption-design">Fig. 4. The binding affinity of control, <i>csgD</i>, and <i>csgA | + | <figcaption class="caption-design">Fig. 4. (A) The formula and graph of the experiment. (The weight of book is 1.6 kilogram, the collision is considered as completely inelastic collision, and the collision time is presumed as 0.1 second.) (B) The binding affinity of control, <i>csgD</i>, and <i>csgA-csgD</i> with given force from different heights.</figcaption> |
</figure> | </figure> | ||
</div> | </div> | ||
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<div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | <div class="d-flex flex-column flex-md-row justify-content-between mb-2"> | ||
<div class="flex-grow-1"> | <div class="flex-grow-1"> | ||
− | <p> As seen in the graph above, <i>csgA</i>-<i>csgD</i> shows an increase in OD<sub>600</sub> value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick <a href="http://parts.igem.org/Part:BBa_K805015" target="_blank" class="linkinthetext">( | + | <p> As seen in the graph above, <i>csgA</i>-<i>csgD</i> shows an increase in OD<sub>600</sub> value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick <a href="http://parts.igem.org/Part:BBa_K805015" target="_blank" class="linkinthetext">(BBa_K805015)</a> by adding <i>csgA</i> and changing the promoter into <i>pLac</i> <a href="http://parts.igem.org/Part:BBa_K3490001" target="_blank" class="linkinthetext">(BBa_K3490001)</a>. |
<br> Please visit <a href="https://2020.igem.org/Team:NCKU_Tainan/Results" target="_blank" class="linkinthetext">Results</a> page for more information. | <br> Please visit <a href="https://2020.igem.org/Team:NCKU_Tainan/Results" target="_blank" class="linkinthetext">Results</a> page for more information. | ||
</p> | </p> | ||
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last=$("body").height()-$(window).height()-35 | last=$("body").height()-$(window).height()-35 | ||
if($(window).scrollTop()>=last){ | if($(window).scrollTop()>=last){ | ||
− | $("#sideNav").css('height', ' | + | $("#sideNav").css('height', '85vh'); |
$("#navtrick").css('top', '0'); | $("#navtrick").css('top', '0'); | ||
− | $(".navrow").css('height', ' | + | $(".navrow").css('height', '4.314vw'); |
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$("#navtrick").css('top', '-4vh'); | $("#navtrick").css('top', '-4vh'); | ||
− | $(".navrow").css('height', ' | + | $(".navrow").css('height', '4.314vw'); |
} | } |
Latest revision as of 22:38, 27 October 2020
Overview
One of the goals we want to achieve is to attach our bacteria to the inner chamber of the contact lens. Here, we used csgD, a master regulator of biofilm production; and csgA, a major subunit of the curli fimbriae. Based on research, curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation[1]. As a transcription factor of curli proteins, csgD can regulate the expression of csgA, leading to biofilm production.
Therefore, we are trying to improve BioBrick BBa_K805015 from the 2017 iGEM TAS-Taipei Team[2] to obtain maximum results of biofilm production and improve the biofilm binding affinity. So, the bacteria will stay attached to the contact lens despite any damages.
We hypothesized that by adding csgA and pLac to the biobrick design, the amount of biofilm produced could be increased. Thus, the overproduction of biofilm enables the bacteria to bind securely to the contact lens. Therefore, we add csgA to the biobrick design and change the promoter into pLac as an improvement.
Experiment Results
Congo Red Assay Experiment
First, we ran SDS-PAGE to identify and quantify the protein expression of CsgA and CsgD. We cultured the bacteria for 2 hours, then added IPTG to induce for 12 hours long, and adjusted the OD600 value to three. As a comparison, we used plasmid that contains pLac-csgD on BW25113, improved parts that include NOS, csgA, and csgD on BW25113, and using PCA24N as control. After that, we transformed into E. coli BL21(DE3) strain. The expected protein size of CsgD is around 24 kDa and CsgA around 17 kDa. The results below have shown the outcome we expected for CsgA and CsgD protein expression.
Next, to prove that our amount of biofilm production increased, we did a test by using congo red[3] dye to observe the curli expression. We compared the absorbance value of BBa_K805015 and BBa_K3490001 to see whether the amount of biofilm production increased or not. If the biofilm amount increases, the color of the solution will appear to be darker. So, after overnight culture, we add congo red dye to all the samples. Then we centrifuge to separate the supernatant and precipitate (pellet). By using a microplate reader, we can measure the absorbance value at 500 nm (congo red) and normalized by 600 nm wavelength which represents the amount of bacteria.
Biofilm Assay Experiment
However, only testing the amount of biofilm production and congo red staining is not enough to fully support our aims that enhance biofilm production and improve the binding affinity of the bacteria. Therefore, we conducted another experiment to compare the binding affinity of bacteria among control, csgD, and csgA-csgD. We presumed that binding affinity is determined by the ability of bacteria to remain attached to surfaces regardless of the external forces applied. Here, we dropped a book from different heights and measuring its OD600 value. By doing so, we can determine the concentration that represents the binding affinity.
As seen in the graph above, csgA-csgD shows an increase in OD600 value when a greater force is given. Hence, we are able to prove that not only enhances the production of biofilm, but our engineered bacteria can also improve its binding affinity. Therefore, we can conclude that we have successfully improved the previous biobrick (BBa_K805015) by adding csgA and changing the promoter into pLac (BBa_K3490001).
Please visit Results page for more information.
References
- Barnhart MM, Chapman MR. Curli Biogenesis and Function. Annual Review of Microbiology. 2006;60(1):131-147.
- Part:BBa K805015 - parts.igem.org. Igem.org. http://parts.igem.org/Part:BBa_K805015. Published 2013. Accessed September 21, 2020.
- Jones CJ, Wozniak DJ. Congo Red Stain Identifies Matrix Overproduction and Is an Indirect Measurement for c-di-GMP in Many Species of Bacteria. c-di-GMP Signaling. 2017:147-156.