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| | <ul class="nav"> | | <ul class="nav"> |
| | <li> | | <li> |
| − | <a href="#PR" class="pageNavBig">PR</a> | + | <a href="#Intro" class="pageNavBig">INTRODUCTION</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#ConDPR" class="pageNavSm">Construct Design</a> | + | <a href="#PR" class="pageNavBig">PROTEORHODOPSIN</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#fTest" class="pageNavSm">Functional Test</a> | + | <a href="#LRI" class="pageNavSm">Ligand-receptor interaction</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#char" class="pageNavSm">Characterization</a> | + | <a href="#PRResults" class="pageNavSm">Results</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#biofilm" class="pageNavBig">BIOFILM</a> | + | <a href="#FDR" class="pageNavSm">Finite Difference Method</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#CanTrap" class="pageNavSm">Can Biofilm trap nanoparticles?</a> | + | <a href="#bio" class="pageNavBig">BIOFILM</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#Reg" class="pageNavSm">How Is Biofilm Production Regulated?</a> | + | <a href="#TR" class="pageNavSm">Trapping Rate</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#ConDBio" class="pageNavSm">Construct Design</a> | + | <a href="#sig" class="pageNavSm">Determining significance</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#Hypo" class="pageNavSm">Hypothesis</a> | + | <a href="#SA" class="pageNavSm">Surface Area</a> |
| | </li> | | </li> |
| | <li> | | <li> |
| − | <a href="#CO" class="pageNavSm">Expression of CsgD and OmpR234 Increases Biofilm Formation</a> | + | <a href="#Bioresults" class="pageNavSm">Results</a> |
| | </li> | | </li> |
| | <li> | | <li> |
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| | <header> | | <header> |
| | <div class="row"> | | <div class="row"> |
| − | <h1 class="name col-lg-12">EXPERIMENTAL SUMMARY</h1> | + | <h1 class="name col-lg-12">MODELING</h1> |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | We use two approaches to capture NPs: 1) target citrate-capped nanoparticles (CC-NPs) using a bacterial membrane protein Proteorhodopsin (PR), and 2) trap a wide variety of NPs using an <i>E. coli</i> biofilm. [pR outline/conclusion] We show experimentally that an <i>E. coli</i> biofilm can trap gold and silver NPs ranging from 15-60 nm. To increase biofilm production, we overexpress CsgD and OmpR234, two positive regulators of biofilm synthesis. We demonstrate that <i>E. coli</i> carrying our constructs produce up to 8 times more biofilm compared to controls. | + | Our model aims to facilitate the implementation of our proteorhodopsin (PR)-expressing bacteria and biofilm prototype in wastewater treatment plants (WWTPs). Using variables which are specific to any WWTP (such as flow rate and water retention time in each tank), we can determine the amount of bacteria or surface area of biofilm needed to reduce nanoparticle concentration in the treated effluent. |
| | </h4> | | </h4> |
| | </div> | | </div> |
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| | </header> | | </header> |
| | <section class="main"> | | <section class="main"> |
| − | <div class="row" id="PR"> | + | <div class="row" id="Intro"> |
| − | <h1 class="col-lg-12 title2">PROTEORHODOPSIN</h1> | + | <h1 class="col-lg-12 title2">INTRODUCTION</h1> |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | Our goal is to remove as many NPs as possible from wastewater systems to minimize damage to the environment and our health. Due to the sheer variety of manufactured NPs and the significant chemical differences between them, we looked for common properties across different types of NPs. Most industrially produced NPs are coated with capping agents to prevent aggregation (ref). <b>We specifically target citrate, the most common NP capping agent</b> (<i>Levard et al.</i> 2012).
| + | Our goal is to remove as many NPs as possible from wastewater systems to minimize damage to the environment and our health. Due to the sheer variety of manufactured NPs and the significant chemical differences between them, we looked for common properties across different types of NPs. Most industrially produced NPs are coated with capping agents to prevent aggregation (ref). <b>We specifically target citrate, the most common NP capping agent</b> (<i>Levard et al.</i> 2012). |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row" id="ConDPR"> | + | <div class="row" id="PR"> |
| − | <h1 class="section-title col-lg-12">Construct Design</h1> | + | <h1 class="title2 col-lg-12">PROTEORHODOPSIN</h1> |
| | </div> | | </div> |
| − | <div class="row"> | + | <div class="row" id="LRI"> |
| − | <h4 class="para col-lg-9">
| + | <h1 class="section-title col-lg-12">Proteorhodopsin and citrate binding modeled as a ligand-receptor interaction</h1> |
| − | PR is a transmembrane protein found in proteobacteria. Since PR has previously been shown to bind citrate through two positively charged lysine residues on its surface (figure 2-1; <i>Béjà et al.</i> 2000; Syed 2011), we hypothesized that PR may also bind citrate capping agents and CC-NPs. <br><br>
| + | |
| − | We obtained the DNA sequence of pR (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). The sequence of pR was then flanked by an upstream strong promoter and strong ribosome combination (BBa_K880005), and a downstream double terminator (BBa_B0015) to maximize expression of PR protein. This final construct (BBa_K2229400; figure 2-2) was ordered from IDT and cloned into pSB1C3, a biobrick backbone.
| + | |
| − | </h4>
| + | |
| − | <div class="image_container col-lg-3">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/8/88/T--TAS_Taipei--figure_2-1-new.png" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b> Figure 2-1. PR-citrate interaction (Syed 2011). </b> Two lysine residues (blue) on the surface of PR can bind citrate (red).</h4>
| + | |
| − | </div>
| + | |
| − | </div><br>
| + | |
| − | <div class="row">
| + | |
| − | <div class="image_container col-lg-6">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/9/9d/T--TAS_Taipei--figure_2-2.png" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b>Figure 2-2. Proteorhodopsin expression.</b> Our construct includes a strong promoter, strong RBS, <i>pR</i> and double terminator.<span class="subCred"> Figure: Justin Y.</span></h4>
| + | |
| − | </div>
| + | |
| − | <div class="image_container col-lg-6">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/a/ad/T--TAS_Taipei--PR.png" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b>Figure 2-3 BBa_K2229450:</b> pR ORF was isolated and cloned into pSB1C3.<span class="subCred"> Figure: Justin Y.</span></h4>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="row" id="fTest">
| + | |
| − | <h1 class="section-title col-lg-12">Functional Test: Does it trap CC-NPs?</h1> | + | |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | blah blah blah <b> bold </b> | + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row" id="char"> | + | <div class="row" id="PRResults"> |
| − | <h1 class="section-title col-lg-12">Characterization: How many NPs can we trap?</h1> | + | <h1 class="section-title col-lg-12">Results of Our Model</h1> |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | blah blah blah <b> bold </b> | + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row" id="biofilm"> | + | <div class="row" id="FDM"> |
| − | <h1 class="col-lg-12 title2">BIOFILM</h1> | + | <h1 class="section-title col-lg-12">Finite-Difference Method (FDM) Explanation</h1> |
| | </div> | | </div> |
| − | <div class="row">
| |
| − | <h4 class="para col-lg-7">
| |
| − | Our biofilm approach was originally inspired by the ability of jellyfish mucus to bioaccumulate gold NPs (AuNPs) and quantum dots through electrostatic interactions (<i>Patwa et al.</i> 2015). A biofilm is a community of microbes embedded in a matrix of extracellular polymeric substances (EPS) consisting of different polysaccharides, proteins, and lipids (Donlan 2002). Recent studies show that the EPS in biofilms can also trap various NPs (<i>Kaoru et al.</i> 2015, <i>Nevius et al.</i> 2012) (figure 3-1).
| |
| − | </h4>
| |
| − | <div class="image_container col-lg-5">
| |
| − | <img src="https://static.igem.org/mediawiki/2017/3/34/T--TAS_Taipei--figure_3-1.jpg" alt="test" id="group">
| |
| − | <h4 class="subtitle"><b>Figure 3-1. We envision using <i>E. coli</i> biofilms (green) to trap nanoparticles of different sizes and composition (pink). </b></h4>
| |
| − | </div>
| |
| − | </div><br>
| |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | Most wastewater treatment plants (WWTPs) already use microbes to break down and remove organic components in wastewater (Sehar & Naz 2016). The recent increase in NP contaminants entering WWTPs, however, poses a threat to these microbes and the treatment process. For example, silver NPs (AgNPs) have antimicrobial effects and other metal oxide NPs can inhibit microbes from performing important processes such as nitrification to remove ammonia (Yun & Lee 2017; Walden & Zhang 2016). <b> Biofilms are nearly four times more resistant to NPs </b> compared to planktonic bacteria, increasing their tolerance to NPs in wastewater (<i>Choi et al.</i> 2010). | + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row" id="CanTrap"> | + | <div class="row" id="bio"> |
| − | <h1 class="section-title col-lg-12">Can Biofilms Trap Nanoparticles?</h1> | + | <h1 class="title2 col-lg-12">BIOFILM</h1> |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | The first step of this approach is to test if an <i>E. coli</i> biofilm can effectively trap NPs. <i>E. coli</i> liquid cultures grown in Luria-Bertani (LB) broth were transferred to petri dishes, each containing glass coverslips to provide a surface for adherence. The dishes were incubated at 37˚C for 7 to 14 days, with 2 mL of LB added every two days to prevent the media from drying out. Viscous, film-like substances began to develop, indicating the production of EPS and biofilm (figure 3-2). At this point, we could extract and wash the biofilm. | + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row"> | + | <div class="row" id="TR"> |
| − | <div class="image_container col-lg-6 col-lg-offset-3">
| + | <h1 class="section-title col-lg-12">Evaluating the trapping rate through the change in substrate concentration and volumetric flow rate</h1> |
| − | <img src="https://static.igem.org/mediawiki/2017/9/9b/T--TAS_Taipei--figure_3-2.jpg" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b>Figure 3-2. Growing <i>E. coli</i> biofilms. </b> A,B) Liquid cultures were plated with glass coverslips and incubated for up to 2 weeks. C) Biofilm can be washed and used. <span class="subCred"> Experiment: Yvonne W.</span></h4>
| + | |
| − | </div>
| + | |
| − | </div> <br>
| + | |
| − | <div class="row">
| + | |
| − | <h4 class="para col-lg-12">
| + | |
| − | Hoping to image the structure of biofilms up close using scanning electron microscopy (SEM), we tried different fixation and preparation methods (figure 3-3 & table 1) (<i>Fischer et al.</i> 2012): 1) freeze drying with a critical point dryer (CPD), 2) fixation with glutaraldehyde (GA), and 3) fixation with a combination of Alcian Blue, GA, and K<sub>4</sub>Fe(CN)<sub>6</sub>. We further improved image quality by taking multiple images of a field of view and stacking the images together to reduce noise (figure 3-4).
| + | |
| − | </h4>
| + | |
| | </div> | | </div> |
| − | <div class="row">
| |
| − | <div class="image_container col-lg-8 col-lg-offset-2" style="padding-bottom: 10px; padding-top: 0px;">
| |
| − | <h4 class="subtitle"><b>Table 1. Comparing different SEM sample preparation protocols. </b>
| |
| − | We tried each method and compiled the advantages and disadvantages.</h4>
| |
| − | <img src="https://static.igem.org/mediawiki/2017/f/fa/T--TAS_Taipei--Table_3-1.PNG" alt="test" id="group">
| |
| − | </div>
| |
| − | </div><br>
| |
| − | <div class="row">
| |
| − | <div class="image_container col-lg-12">
| |
| − | <img src="https://static.igem.org/mediawiki/2017/e/e0/T--TAS_Taipei--figure_3-3-min.jpg" alt="test" id="group">
| |
| − | <h4 class="subtitle"><b>Figure 3-3. Comparing different SEM sample preparation protocols. </b> <i>E. coli</i> samples were prepared for SEM. A) Critical Point Drying seems to change cell morphology, whereas B,C) fixation with GA preserved both cell shape and biofilm structure. <span class="subCred"> SEM Imaging & Figure: Justin Y.</span>
| |
| − | </h4>
| |
| − | </div>
| |
| − | </div><br>
| |
| − | <div class="row">
| |
| − | <div class="image_container col-lg-10 col-lg-offset-1">
| |
| − | <img src="https://static.igem.org/mediawiki/2017/e/eb/T--TAS_Taipei--figure_3-4-min.jpg" alt="test" id="group">
| |
| − | <h4 class="subtitle"><b>Figure 3-4. Image stacking decreases noise.</b> Here, 26 images of the same field of view were taken and stacked using Adobe Photoshop.<span class="subCred"> SEM Imaging & Figure: Justin Y.</span></h4>
| |
| − | </div>
| |
| − | </div><br>
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| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | To test if biofilms can trap NPs, we used a solution containing 30 nm AuNPs (from Sigma Aldrich). Because the AuNP solution is purple in color, we can take absorbance measurements. Four experimental groups were set up (figure 3-5): a negative control containing only AuNPs, and three tubes containing AuNP with either planktonic bacteria, biofilm, or “dead” biofilm (treated with antibiotics). The four groups were shaken for 24 hours and centrifuged to isolate the supernatant (figure 3-5, C). The supernatant contains free AuNPs, which were measured using a spectrophotometer at 527 nm. | + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row"> | + | <div class="row" id="sig"> |
| − | <div class="image_container col-lg-10 col-lg-offset-1">
| + | <h1 class="section-title col-lg-12">Determining the significance of different factors</h1> |
| − | <img src="https://static.igem.org/mediawiki/2017/3/38/T--TAS_Taipei--figure_3-5.jpg" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b>Figure 3-5. Biofilms trap nanoparticles </b> A,B) Experimental setup. C,D) Gold nanoparticles (AuNPs) were incubated with either bacteria or biofilm for 24 hours, and then centrifuged. Free AuNPs were expected to remain in the supernatant. Shown are representative images and graphs.<span class="subCred"> Experiment: Yvonne W. Figure: Justin Y.</span></h4>
| + | |
| − | </div>
| + | |
| − | </div><br>
| + | |
| − | <div class="row">
| + | |
| − | <h4 class="para col-lg-12">
| + | |
| − | The negative control and the AuNP with planktonic bacteria groups did not change the purple color of the AuNP solution, indicating that bacteria alone cannot trap NPs (figure 3-5, C and D). The addition of biofilm, however, greatly reduced the amount of AuNP in the supernatant. Even with antibiotics, AuNP levels in the supernatant were still reduced, suggesting that the removal of AuNPs depends on the sticky and slimy extracellular components of biofilm and not on the bacteria itself. When we fixed and imaged the biofilm + AuNP sample by SEM (figure 3-6), we could see NPs in EPS areas. This confirmed our idea that NPs were being trapped in the EPS layer of biofilms.
| + | |
| − | </h4>
| + | |
| | </div> | | </div> |
| − | <div class="row">
| |
| − | <div class="image_container col-lg-6 col-lg-offset-3">
| |
| − | <img src="https://static.igem.org/mediawiki/2017/a/a5/T--TAS_Taipei--figure_3-6.jpg" alt="test" id="group">
| |
| − | <h4 class="subtitle"><b>Figure 3-6. SEM Image showing AuNPs trapped by biofilm.</b> A biofilm+AuNP sample was fixed with GA. Some EPS is preserved (red) and AuNPs (white) seemed to aggregate and adhere onto the EPS.<span class="subCred"> SEM Imaging: Justin Y.</span></h4>
| |
| − | </div>
| |
| − | </div><br>
| |
| − | <div class="row" id="Reg">
| |
| − | <h1 class="section-title col-lg-12">How Is Biofilm Production Regulated?</h1>
| |
| − | </div>
| |
| − | <div class="row">
| |
| − | <div class="image_container col-lg-8 col-lg-offset-2">
| |
| − | <img src="https://static.igem.org/mediawiki/2017/c/ce/T--TAS_Taipei--figure_3-7.jpg" alt="test" id="group">
| |
| − | <h4 class="subtitle"><b>Figure 3-7. Two curli operons—<i>csgBA</i> and <i>csgDEFG</i>—direct biofilm synthesis.</b><span class="subCred"> Figure: Justin Y.</span></h4>
| |
| − | </div>
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| − | </div><br>
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| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | <b>In <i>E. coli</i>, biofilm synthesis is mainly mediated through two curli operons</b> (Barnhart & Chapman 2006). Curli fibers facilitate cell-surface and cell-cell adhesion, biofilm synthesis, and are the main protein components of the EPS (<i>Reichhardt et al.</i> 2015, Barnhart & Chapman 2006). The operons (<i>csgBA</i> and <i>csgDEFG</i>) control the expression of six proteins essential to biofilm formation (figure 3-7). CsgA and CsgB are curli monomers which can polymerise to form curli fibers. <b>CsgD is an activator of <i>csgBA</i> transcription</b> (figure 3-7). CsgE, CsgF, and CsgG help export CsgA and CsgB out of the cell. <b>The operon <i>csgDEFG</i> can be activated by the protein OmpR</b>, and the subsequent expression of all six proteins increase biofilm formation (Barnhart & Chapman 2006).
| + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row" id="ConDBio"> | + | <div class="row" id="SA"> |
| − | <h1 class="section-title col-lg-12">Construct Design</h1> | + | <h1 class="section-title col-lg-12">Surface Area</h1> |
| | </div> | | </div> |
| | <div class="row"> | | <div class="row"> |
| | <h4 class="para col-lg-12"> | | <h4 class="para col-lg-12"> |
| − | <b>Three constructs were built to upregulate curli production</b> by overexpressing CsgD, OmpR234 (a mutant form of OmpR which is constitutively active), or both. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), <i>csgD</i> (BBa_K805015), <i>ompR234</i> (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.
| + | Lorem ipsum dolor sit amet, consectetur adipisicing elit. Est sit odit dolorum illum nemo harum voluptatem sapiente consequuntur hic, earum quos facilis ea magnam, laudantium ipsam. Inventore labore non, accusantium nemo? Similique optio praesentium ad aliquam illo, ipsum, accusantium explicabo architecto molestias fugiat maiores. Optio pariatur, aliquam quia commodi dignissimos? |
| | </h4> | | </h4> |
| | </div> | | </div> |
| − | <div class="row"> | + | <div class="row" id="BioResults"> |
| − | <div class="image_container col-lg-6">
| + | <h1 class="section-title col-lg-12">Biofilm time trial results, finding average trapping rate (per SA)</h1> |
| − | <img src="https://static.igem.org/mediawiki/2017/3/33/T--TAS_Taipei--figure_3-8.jpg" alt="test" id="group">
| + | |
| − | <h4 class="subtitle"><b> Figure 3-8. CsgD expression. </b> Our construct includes a strong promoter, strong RBS, csgD and double terminator.<span class="subCred"> Figure: Justin Y.</span></h4>
| + | |
| − | </div>
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| − | <div class="image_container col-lg-6">
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| − | <img src="https://static.igem.org/mediawiki/2017/b/b3/T--TAS_Taipei--figure_3-9.jpg" alt="test" id="group">
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| − | <h4 class="subtitle"><b> Figure 3-9. OmpR234 Expression </b> Our construct includes a strong promoter, strong RBS, ompR234 and double terminator.<span class="subCred"> Figure: Justin Y.</span></h4>
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| − | For strong CsgD expression (figure 3-8, BBa_K2229100), <i>csgD</i> was inserted behind BBa_K880005 (figure 3-10, BBa_S05397), and then before BBa_B0015 (figure 3-11).
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| − | For strong OmpR234 expression (figure 3-9, BBa_K2229200), <i>ompR234</i> was inserted before BBa_B0015 (figure 3-10, BBa_S05398) and then behind BBa_K880005 (figure 3-11).
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| − | <h4 class="subtitle"><b>Figure 3-10. PCR Check for BBa_K880005 +CsgD and OmpR234 +BBa_B0015. </b> The expected size of BBa_K880005 +CsgD is 1000 bp (orange box) and OmpR234 +BBa_B0015 is 1100 bp (blue box).<span class="subCred"> Cloning: Catherine Y., Dylan L., Justin Y.</span></h4>
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| − | <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-11.jpg" alt="test" id="group">
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| − | <h4 class="subtitle"><b> Figure 3-11. PCR Check for BBa_K2229100 & BBa_K2229200. </b> The expected size of BBa_K2229100 (CsgD full construct) is 1100 bp (orange box), and BBa_K2229200 (OmpR234 full construct) is 1200 bp (blue box).<span class="subCred"> Cloning: Catherine Y., Dylan L., Justin Y.</span></h4>
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| − | <h4 class="subtitle"><b>Figure 3-12. CsgD and OmpR234 Expression </b> Our construct includes a strong promoter, two strong RBS, <i>csgD</i>, <i>ompR234</i> and double terminator.<span class="subCred"> Figure: Justin Y.</span></h4>
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| − | To strongly express both CsgD and OmpR234 (figure 3-12, BBa_K2229300), a strong RBS (BBa_B0034) was inserted in front of the intermediate BBa_S05398 (<i>ompR234</i>+double terminator) to make BBa_S05399 (RBS+<i>ompR234</i>+double terminator). FInally, BBa_S05397 (K880005+csgD) was inserted before BBa_S05399 to complete the full construct (BBa_K2229300) (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.
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| − | <h4 class="subtitle"><b>Figure 3-13. PCR Check for BBa_K2229300. </b> The expected size of BBa_K2229300 is 1900 bp (green box)<span class="subCred"> Cloning: Catherine Y., Dylan L., Justin Y.</span></h4>
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| − | <h1 class="section-title col-lg-12">Hypothesis</h1> | + | |
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| − | <h4 class="subtitle"><b>Figure 3-14. Overexpression of CsgD and/or OmpR234 upregulates the curli operon to different degrees </b> We hypothesize that biofilm production would be upregulated (in increasing order) if we overexpress A) CsgD, B) OmpR234, or C) both.<span class="subCred"> Figure: Justin Y.</span></h4>
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| − | <b>We hypothesized that biofilm production would be upregulated (in increasing order) if we overexpress CsgD, OmpR234, or both</b> (figure 3-14). Overexpression of CsgD would result in more curli monomers, but no transport proteins to carry the monomers out of the cell. Overexpression of OmpR234 would allow curli monomers to be exported and form fibers and biofilm. Finally, when both CsgD and OmpR234 are overexpressed, twice the amount of curli monomers should be made and exported to form fibers and biofilm.
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| − | <h1 class="section-title col-lg-12">Expression of CsgD and OmpR234 Increases Biofilm Formation</h1>
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| − | To test the expression of CsgD and OmpR234, we ran SDS-PAGE using transformed and lysed <i>E. coli</i> cultures (figure 3-15). Cultures transformed with the basic parts BBa_K805015 (csgD ORF alone) and BBa_K342003 (<i>ompR234</i> ORF alone) were used as negative controls. We expected to see CsgD around 25 kDa and OmpR234 around 27 kDa (<i>Brombacher et al.</i> 2006; Martinez & Stock 1997). Compared to negative controls, <b>thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) and BBa_K2229200 (OmpR234 overexpression)</b> (figure 3-15; proteins bands are marked by asterisks).
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| − | In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (<i>Robinson et al.</i> 2006; <i>Uhlich et al.</i> 2009; <i>Shu et al.</i> 2012) . This suggests that, as expected, <b>BBa_K2229300 stimulates the production of all curli proteins</b> (predicted proteins and sizes are labeled in figure 3-15).
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| − | <h4 class="subtitle"><b>Figure 3-15. SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively.</b> Predicted proteins from the curli operons are listed on the right, and <i>E. coli</i> expressing GFP was used as a positive control.</b><span class="subCred"> Protein Gel: & Figure: Justin Y.</span></h4>
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| − | After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips, and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-16, 3-17, 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
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| − | We find that <b>overexpressing CsgD and/or OmpR234 increases biofilm production to different degrees</b>, as we hypothesized (figure 3-19). Overexpression of CsgD (BBa_K2229100) doubles biofilm production compared to the negative control BBa_K805015 (figure 3-16); overexpression of OmpR234 (BBa_K2229200) leads to about 8 times more biofilm compared to the negative control BBa_K342003 (figure 3-17 & figure 3-18, A). CGU_Taiwan helped us independently verify our OmpR234 overexpression results using a different dye, crystal violet, which is also commonly used to stain biofilms (figure 3-18, B). Interestingly, both biofilms characterized in our assay are found <i>around</i> the glass coverslip and do not seem to stick well to the glass surface (figures 3-16 & 3-17).
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| − | <h4 class="subtitle"><b> Figure 3-16: Overexpression of CsgD (BBa_K2229100) doubles biofilm production </b> A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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| − | <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-17.jpg" alt="test" id="group">
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| − | <h4 class="subtitle"><b> Figure 3-17: Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control </b> A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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| − | <img src="https://static.igem.org/mediawiki/2017/7/70/T--TAS_Taipei--figure_3-18.jpg" alt="test" id="group">
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| − | <h4 class="subtitle"><b> Figure 3-18 Overexpression of OmpR234. </b> A) OmpR234 overexpression (BBa_K2229200) produces more biofilms. B) CGU_Taiwan independently verified that BBa_K2229200 increases biofilm production through crystal violet staining.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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| − | When all three constructs were compared, we find that overexpression of both OmpR234 and CsgD (BBa_K2229300) increased biofilm production the most (figure 3-19). BBa_K2229300 also increased adhesion to our glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps (figure 3-19, A).
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| − | <img src="https://static.igem.org/mediawiki/2017/4/4c/T--TAS_Taipei--figure_3-19.jpg" alt="test" id="group">
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| − | <h4 class="subtitle"><b> Figure 3-19 Overexpression of both CsgD and OmpR234 (BBa_K2229300) increases biofilm production the most. </b> A) Congo red assay stains biofilms. BBa_K2229300 increases adhesion to glass surfaces. B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.<span class="subCred"> Experiment & Figure: Yvonne W.</span></h4>
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| − | In summary, <b>we demonstrate that biofilms can trap NPs</b> and our constructs function as hypothesized. <b>Our collection of constructs (BBa_K2229100, BBa_K2229200, and BBa_K2229300) can successfully upregulate biofilm production to varying degrees.</b>
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| − | *Details on any experimental setup can be found in the <a href="https://2017.igem.org/Team:TAS_Taipei/Notebook">Protocols</a> section of our lab notebook.
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| − | <div class="row" id="Ref">
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| − | <h1 class="col-lg-12 title2">REFERENCES</h1>
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| − | William Chen - the best
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| | </div> | | </div> |
| | </section> | | </section> |
