q

wiki/pages/engineering.html

@@ -305,18 +305,21 @@ endblock %}
       <h6><span>First attempt</span></h6>
       <p><span>We collected the cell media of the cancer cells containing transfected plasmids, which were then
           photographed with an inverted microscope. At first, we recorded a bright image with a lot of light-emitting
-          spots. However, it was suspected to be a false positive result, as the spots are larger than the cells
+          spots. However, it was suspected to be a false positive result, and the reasons are evaluated below.
           themselves.</span></p>
 
       <h6><span>First attempt evaluation</span></h6>
       <p><span>We have discussed the results with Dr Yu, who has extensive experience in GFP analysis. (For more, please
           refer to our </span><a href="human-practices">integrated human practice</a><span> page)</span></p>
-      <p><span>Possible reasons:</span></p>
+      <p><span>Possible reasons for the false positive result:</span></p>
+      <ol>
+        <li><span>Most of the spots are a result of </span><strong>autofluorescence </strong><span>from the cell
+            organelles but not GFP.</span></li>
+        <li><span>Only the cell media was photographed, which mostly consisted of </span><strong>dead cells that could
+            not transcribe genes.</strong></li>
+      </ol>
+      <p><span>Possible reasons why fluorescence from GFP is not observed:</span></p>
       <ol>
-        <li><span>Most of the spots are a result of </span><strong>autofluorescence </strong><span>from the cells
-            themselves.</span></li>
-        <li><span>The cell media was photographed, which mostly consisted of </span><strong>dead cells that cannot
-            transcribe genes.</strong></li>
         <li><span>There was a noticeable delay between collecting the supernatant and photographing, which may lead to
             the </span><strong>degradation of GFP.</strong></li>
         <li><span>The </span><strong>concentrations </strong><span>of plasmids/cells may be</span><strong> too
@@ -329,9 +332,10 @@ endblock %}
       <p><span>After evaluating the previous attempt, we made the following changes:</span></p>
       <ol>
         <li><span>Photograph cells directly instead of cell media</span></li>
-        <li><span>Cells were kept in a 37&deg;C incubator with 5% CO2 until minutes before photographing</span></li>
+        <li><span>Cells were kept in a 37&deg;C incubator with 5% CO2 until minutes before photographing to prevent the
+            degradation of GFP.</span></li>
         <li><span>Cells were photographed in the same position with and without the blue light filter to confirm the
-            luminescence given out by actual cells.</span></li>
+            fluorescence given out by actual cells.</span></li>
       </ol>
       <p><span>We have successfully photographed the fluorescence given out by GFP genes transcribed by
           pENTR1A-PSMA-GFP.</span></p>
@@ -362,7 +366,8 @@ endblock %}
         <li><span>Photographs must be done </span><strong>rapidly</strong><span> after the cells are taken out of the
             incubator</span></li>
       </ol>
-      <p><span>Moreover, we confirmed the success of converting from a protein level to a genetic level. Plasmid
+      <p><span>Moreover, we confirmed the success of converting from a protein level to a genetic level. Unlike protein
+          synthesis, plasmid
           constructions </span><strong>are easily replicable</strong><span>, and we were able to </span><strong>not only
           discover issues earlier but also synthesise a high concentration of plasmids</strong><span>. By
           purchasing:</span></p>
@@ -425,14 +430,17 @@ endblock %}
 
       <a name="build2"></a>
       <h5><span>Build</span></h5>
+      <h6><span>Plasmid construction</span></h6>
       <p><span>We purchased the pENTR1A backbone along with another plasmid that contains a PSMA-Gluc gene.</span></p>
       <p><span>After digesting both plasmids, we performed gel electrophoresis. We then purified the agarose gel and
           ligated the two genes to create the plasmid pENTR1A-PSMA-Gluc (for more details, see cycle 3). The ligated
           plasmids were transformed into competent DH5&alpha; cells and subsequently extracted. To confirm the correct
-          gene length, we digested the extracted plasmids. Following this, we transfected various concentrations of the
+          gene length, we digested the extracted plasmids. </span></p>
+      <h6><span>Plasmid construction</span></h6>
+      <p><span>Following this, we transfected various concentrations of the
           plasmids into different amounts of the PSMA-positive cancer cell line MLLB-2, which were subcultured into
           96-well plates one night beforehand. The cell lines are incubated for 2 more days in a 37&deg;C environment
-          with 5% CO</span><span>2</span><span>.</span></p>
+          with 5% CO2.</span></p>
       <table>
         <tbody>
           <tr>
@@ -473,10 +481,11 @@ endblock %}
       <h6><span>Attempt 2</span></h6>
       <p><span>We fixed the problem from the previous attempt and measured luminescence at another setting.</span></p>
       <img src="https://static.igem.wiki/teams/5441/cycle/c2f4.jpg">
-      <p><span>Settings for the measurement from attempt 2</span></p>
+      <p><span>Settings for the measurement from attempt 2. Blanks are set to wells with only cell media without any
+          plasmids.</span></p>
 
       <h6><span>Attempt 2: Evaluation</span></h6>
-      <p><em><span>insert our results</span></em></p>
+      <img src="https://static.igem.wiki/teams/5441/parts/gluc-results-line.jpg">
       <p><span>100,000 cells/well</span></p>
       <p><span>At 100k cells per well, change in plasmid concentration did not show significant differences for
           luminescence measured. Moreover, the luminescence measured is lower than the blank samples, which shows signs