5639635

wiki/pages/contribution.html

@@ -25,7 +25,7 @@
       <img src='https://static.igem.wiki/teams/4325/wiki/page/contribution/figure-1.png' class='figure-img img-fluid rounded' alt='...'>
       <figcaption class='figure-caption text-center'>Figure 1: Colony growth of <i>E. coli</i> TOP10 containing pDawn-RBSNNN-X174E-rrnB T1 (a) in the dark and (b) under blue light.</figcaption>
     </figure>
-    <p>We performed a new round of phenotyping assay for pDawn-RBSNNN-X174E-rrnB T1 (<a title="BBa_K3740044" href="http://parts.igem.org/Part:BBa_K3740044" target="_blank" rel="noopener">BBa_K3740044</a>). First, the monoclonal transformants of <em>E. coli </em>TOP10 with pDawn-RBSNNN-X174E-rrnB T1 were picked and grown on two parallel new LB plates, with one cultured in the dark and the other under blue light respectively to screen for correct builds. As shown in Figure 1, pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 had a total of 10 strains which survived in the dark but failed to grow under blue light. This suggests that the blue light induced lysis system can function as intended in <em>E. coli</em> Top10.</p>
+    <p>We performed a new round of phenotyping assay for pDawn-RBSNNN-X174E-rrnB T1 (<a title="BBa_K3740044" href="http://parts.igem.org/Part:BBa_K3740044" target="_blank" rel="noopener">BBa_K3740044</a>). First, the monoclonal transformants of <em>E. coli </em>TOP10 with pDawn-RBSNNN-X174E-rrnB T1 were picked and grown on two parallel new LB plates, with one cultured in the dark and the other under blue light respectively to screen for correct builds. As shown in Figure 1, pSEVA331-pDawn-RBSNNN-X174E-rrnB T1 had a total of 10 strains which survived in the dark but failed to grow under blue light. This suggests that the blue light induced lysis system can function as intended in <em>E. coli</em> TOP10.</p>
     <p>From the above successful recombinant candidates (red squares in Figure 1), two bacterial strains were picked and grown in two parallel tubes of LB medium respectively, with one tube in the dark and the other tube under blue light to track. The OD<sub>600</sub><sub> </sub>values of the two strains cultured under the two different illumination conditions were measured at hourly intervals. As shown in Figure 2, both strains grew normally in the dark. For the #1 strain, the OD<sub>600</sub> value increased during the first four hours of blue light illumination and then declined significantly. This delay in lysis indicates it takes time for the lysis protein X174E to accumulate to lethal concentration under blue light. That is to say, the blue light sensitive lysis system of this strain is not efficient enough. For the #11 strain, the OD<sub>600</sub> value remained low during the first five hours of blue light illumination indicating the effectiveness of the system, but then increased dramatically implying that loss-of-function mutations could occurred in these genes or in the host genome and that the favorable mutant overtakes the parental population. Therefore, the blue light sensitive lysis system of this strain is not evolutionarily stable.</p>
     <figure class='figure imgbox center-block'>
       <img src='https://static.igem.wiki/teams/4325/wiki/page/contribution/figure-2.png' class='figure-img img-fluid rounded' alt='...'>

wiki/pages/experiments.html

@@ -222,8 +222,8 @@ th {
         <span>6.8. After resuspending with 5mL of 15% glycerol, place the bacteria solution in a centrifuge at 4℃ for 5min at 4100rpm. Discard the supernatant solution.</span>
         <span>6.9. After resuspending the bacteria with 1mL of 15% pre-cooled glycerol, pack each 100&mu;L of it individually into 1.5mL centrifuge tubes.</span>
         <span>* Note: After freezing, the transform efficiency of the competent cell may decrease, so immediately use may yield the highest efficiency.</span>
-        <span>6.10. Add 600ng plasmid to receptor cells and flick gently, then draw them into a (0.1cm) electroporation cup and set 3000V and 5-8ms. After completion, immediately take the bacteria to 500mL HS medium and Cellulose, and culture it at 230rpm and 30℃ for 16h. Calculation: the volume of plasmid=600ng/Concentration (no more than 10&mu;L.)</span>
-        <span>6.11. After centrifuging for 1min, resuspend with 100&mu;L of medium and spread the HS Agar (including antibiotics), the colony will grow in patches for 24-72h. It needs to be identified by PCR to ensure the correct plasmid.&emsp;</span></p>
+        <span>6.10. Add 600ng plasmid to competent cells and flick gently, then transfer the bacteria solution into a (0.1cm) electroporation cup and set 3000V for 5-8ms. After completion, immediately take the bacteria to 800mL HS medium with 0.2%(v/v) Cellulose, and culture it at 220rpm and 30℃ for 16h. <br>Calculation: the volume of plasmid=600ng/Concentration (no more than 10&mu;L.)</span>
+        <span>6.11. After centrifuging for 10min, discard 700μL surpernatant solution resuspend with 100&mu;L of HS medium and spread the bacteria solution on the plate with the HS agar (including antibiotic), the colonies will grow for 24-72h. It needs to be verified by PCR to ensure the transform results.&emsp;</span></p>
 
         <h2 class='animate wiki-title2' data-animate='zoomIn'>GSH concentration test module</h2>
         <h3 class='animate wiki-title3'>Materials</h3>
@@ -236,7 +236,7 @@ th {
           <ul>
             <li>DMSO</li>
             <li>GSH Scavenging Adjuvant</li>
-            <li>GSH ScavengingReagent</li>
+            <li>GSH Scavenging Reagent</li>
             </ul>
           </div>
           <h3 class='animate wiki-title3'>Procedure</h3>

wiki/pages/implementation.html

@@ -20,7 +20,7 @@
   <div class="col-lg-9">
     <div id="postContentDiv" class="span9">
       <h2 class='animate wiki-title2' data-animate='zoomIn'>Overview</h2>
-      <p>The goal of our project is to construct a bacterial strain "<i>Beauty G. hansenii</i>" (See  <a href="https://2022.igem.wiki/szpt-china/description" target="_blank">Description</a> for details) with synthetic biology and optogentic approaches and used it to develop a live bacterial skincare product with multiple efficacy. Under near-infrared light, the engineered bacteria can produce bacterial cellulose (BC) for moisturizing. Under blue light, they can release glutathione (GSH) as well as bacterial lysate to provide antioxidative effect and maintain skin microbiome. Our product is <b> presented in the form of live bacteria,</b> committed to providing users with multiple benefits of moisturizing, anti-spots, brightening, anti-aging, repairing, and skin microbiome maintenance as well as a brand-new skincare experience.
+      <p>The goal of our project is to construct a bacterial strain "<i>Beauty G. hansenii</i>" (See  <a href="https://2022.igem.wiki/szpt-china/description" target="_blank">Description</a> for details) with synthetic biology and optogentic approaches and use it to develop a live bacterial skincare product with multiple efficacy. Under near-infrared light, the engineered bacteria can produce bacterial cellulose (BC) for moisturizing. Under blue light, they can release glutathione (GSH) as well as bacterial lysate to provide antioxidative effect and maintain skin microbiome. Our product is <b> presented in the form of live bacteria,</b> committed to providing users with multiple benefits of moisturizing, anti-spots, brightening, anti-aging, repairing, and skin microbiome maintenance as well as a brand-new skincare experience.
       </p>
       <h2 class='animate wiki-title2' data-animate='zoomIn'>
         Users

wiki/pages/improve.html

@@ -21,7 +21,7 @@
           <img src='https://static.igem.wiki/teams/4325/wiki/page/design/project4.jpeg' class='figure-img img-fluid rounded' alt='...'>
           <figcaption class='figure-caption text-center'>Figure 1: The genetic circuit of the lysis and safety module</figcaption>
         </figure>
-      <p>The blue light response part pDawn is used to control the expression of lysis genes and characterized in the resultant system (Figure 1). The host chassis expressing this system is expected to grow normally without blue light and to lyse under blue light. However, we found that a significant proportion of the recombinant colonies did not grow very well in the dark, indicating a leak expression of the lysis genes (Figure 2-a). The colony which grew normally in the dark was picked and grown to logarithmic growth phase. But the strain continued growing under blue light, which means that the system became disabled in rapidly growing cells (Figure 2-b). It is suggestd that the above mentioned leaky expression of the lysis genes leads to loss-of-function mutation and that the favorable mutant overtakes the parental population.</p>
+      <p>The blue light response part pDawn is used to control the expression of lysis genes and characterized in the resultant system (Figure 1). The host chassis expressing this system is expected to grow normally without blue light and to lyse under blue light. However, we found that there is a significant proportion of the recombinant colonies did not grow very well in the dark, indicating a leak expression of the lysis genes (Figure 2-a). The colony which grew normally in the dark was picked and grown to logarithmic growth phase. But the strain continued growing under blue light, which means that the system became disabled in rapidly growing cells (Figure 2-b). It is suggestd that the above mentioned leaky expression of the lysis genes leads to loss-of-function mutation and that the favorable mutant overtakes the parental population.</p>
       <figure class='figure imgbox center-block'>
         <img src='https://static.igem.wiki/teams/4325/wiki/page/improvement/improvement2.jpg' class='figure-img img-fluid rounded' alt='...'>
         <figcaption class='figure-caption text-center'>Figure 2: (a) Colony growth of <i>E. coli</i> TOP10 containing pDawn(cI-LVA)-RBS070-LKD in the dark<br>(b) Growth curve of <i>E. coli</i> TOP10 containing pDawn(cI-LVA)-RBS070-LKD</figcaption>

wiki/pages/results.html

@@ -26,7 +26,7 @@
   <div class='col-lg-9'>
     <div id='postContentDiv' class='span9'>
     <h2 class='animate wiki-title2' data-animate='zoomIn'>Engineering of the GshF expression and GSH production module</h2>
-    <p>The goal of this module is to construct an expression system of the bifunctional glutathione synthetase GshF, which can be employed to enhance glutathione production in <em>G. hansenii</em>. To this end, the gene <em>gshF</em> was firstly optimized regarding the codon sequence, and then ligated to pSEVA331, which was finally introduced into <em>E. coli </em>Top10 to obtain the expression system. To the DNA constructs, specific primers were designed to amplify a partial region of <em>gshF</em>, and the size of the PCR product met with our expectation (Figure 1a). In addition, to examine the efficacy of the expression system, the molecular weight of the HiS-tag-labelled GshF protein was checked using western blot assay (Figure 1b). Interestingly, even in the genetic background of <em>E. coli</em> TOP10, GshF is active and functional since the GSH production has been significantly increased in TOP10 following transformation with the pSEVA311-based <em>gshF</em> expression system (Figure 1c).</p>
+    <p>The goal of this module is to construct an expression system of the bifunctional glutathione synthetase GshF, which can be employed to enhance glutathione production in <em>G. hansenii</em>. To this end, the gene <em>gshF</em> was firstly optimized regarding the codon sequence, and then ligated to pSEVA331, which was finally introduced into <em>E. coli </em>TOP10 to obtain the expression system. To the DNA constructs, specific primers were designed to amplify a partial region of <em>gshF</em>, and the size of the PCR product met with our expectation (Figure 1a). In addition, to examine the efficacy of the expression system, the molecular weight of the HiS-tag-labelled GshF protein was checked using western blot assay (Figure 1b). Interestingly, even in the genetic background of <em>E. coli</em> TOP10, GshF is active and functional since the GSH production has been significantly increased in TOP10 following transformation with the pSEVA311-based <em>gshF</em> expression system (Figure 1c).</p>
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         <div class='more-img'>
           <img src='https://static.igem.wiki/teams/4325/wiki/page/results/figure1.png' class='imgbox-img' alt='...' style="max-width: none;width:80%;max-height: 500px;">
@@ -121,7 +121,7 @@
             </div>
             <figcaption class='figure-caption text-center' >Figure 7: (a) Colony growth of <i>E. coli</i> TOP10 containing BX03 (a1) in the dark, (a2) under blue light; <br>(b) Colony growth of <i>E. coli</i> TOP10 containing BX04 (b1) in the dark, (b2) under blue light; <br>(c) Colony growth of <i>E. coli</i> TOP10 containing BL03 (c1) in the dark, (c2) under blue light; <br>(d) Colony growth of <i>E. coli</i> TOP10 containing BL04 (d1) in the dark, (d2) under blue light.</figcaption>
           </div>
-          <p>The kill switch variants BX03, BX04, BL03 and BL04 were therefore generated either by deleting the degradation tag LVA in the original repressor cI of pDawn or adding the degradation tag LVA downstream the lysis gene. The plasmids were introduced into <i>E. coli</i> Top10 by electroporation to obtain the transformants (Table 1). Then, the <i>E. coli</i> strains that grew in the dark but not under blue light were selected (BX03, BL03, BL04 in Figure 7) and cultured in LB medium in the dark until the OD600 reached around 0.6. Finally, the bacterial growth was further monitored under blue light. We found that those three new engineered strains showed decreased OD600 values in response to blue light (Figure 8), indicating the effective refinement of the inducible suicide plasmid in rapid growing cells.</p>
+          <p>The kill switch variants BX03, BX04, BL03 and BL04 were therefore generated either by deleting the degradation tag LVA in the original repressor cI of pDawn or adding the degradation tag LVA downstream the lysis gene. The plasmids were introduced into <i>E. coli</i> TOP10 by electroporation to obtain the transformants (Table 1). Then, the <i>E. coli</i> strains that grew in the dark but not under blue light were selected (BX03, BL03, BL04 in Figure 7) and cultured in LB medium in the dark until the OD<sub>600</sub> reached around 0.6. Finally, the bacterial growth was further monitored under blue light. We found that those three new engineered strains showed decreased OD<sub>600</sub> values in response to blue light (Figure 8), indicating the effective refinement of the inducible suicide plasmid in rapid growing cells.</p>
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             <div class='more-img'>
               <img src='https://static.igem.wiki/teams/4325/wiki/page/results/figure8.png' class='imgbox-img' alt='...' style="max-width: none;width:90%;max-height: 500px;">