Team:HKUST-Hong Kong/Future Work

From 2012.igem.org

(Difference between revisions)
(GFP coding gene needs not italicized)
 
(10 intermediate revisions not shown)
Line 376: Line 376:
               <h1></h1>
               <h1></h1>
           </div>
           </div>
-
           <p>Three-month wet lab work is not enough to complete our project and the result we obtained is not conclusive enough to demonstrate the full story of our project. There is still a lot characterization methods need to be kept on testing and a lot of experiments need to be repeated in order to obtain significant data. </p>
+
           <p>Unfortunately, our wet lab only lasted for three months and we were short of time. The completeness of our project was heavily deterred. Inconclusive results from some of the modules have also increased difficulty in demonstrating the full story of B. hercules. Many characterization methods have yet to be attempted. Some experiments could be repeatd as well, and hopefully that would increase confidence in previously obtaiend data. </p>
 +
<p>If more time is allowed, the following would be what we wish to pursue:</p>
 +
 
<ol>
<ol>
-
   <li>Finish assembly of the parts from three modules into pDG1661 and transformed into <i>Bacillus subtilis</i>: so far, we have been inserted BMP2 construct (<a href="http://partsregistry.org/Part:BBa_K733016" target="_blank">BBa_K733016</a> and <a href="http://partsregistry.org/Part:BBa_K733017" target="_blank">BBa_K733017</a>) and RPMrel construct (<a href="http://partsregistry.org/Part:BBa_K733007" target="_blank">BBa_K733007</a>) into vector pDG1661 separately for characterization. The next step is to assemble parts from all three modules into pDG1661, generating our final construct and move on to final characterization.</li>
+
   <li><strong>Finish assembly of the parts from the three modules into pDG1661 and transformed into <i>Bacillus subtilis</i>:</strong> <br/> So far, we have inserted the BMP2 construct (<a href="http://partsregistry.org/Part:BBa_K733016" target="_blank">BBa_K733016</a> and <a href="http://partsregistry.org/Part:BBa_K733017" target="_blank">BBa_K733017</a>) and the RPMrel construct (<a href="http://partsregistry.org/Part:BBa_K733007" target="_blank">BBa_K733007</a>) into the integration vector pDG1661 separately for characterization. The next step would be to piece these separated parts, as well as the remaining constructs, together to complete our B. hercules. Then we could move on to the final characterization of the strain.</li>
-
   <li>Continue characterizing the binding ability of B. hercules to colon adenocarcinoma HT-29:
+
   <li><strong>Continue the characterization on binding capability of B. hercules to colon adenocarcinoma HT-29:</strong> <br/>
-
Originally we plan to co-culture B. hercules with HT-29 cell (colon adenocarcinoma) and HBE16 (Human bronchial epithelial cell) and detect the attached <i>B. subtilis</i> through its fluorescence signal. However, when we finished our construct, we find out that no GFP signal can be detected in <i>B. subtilis</i>. One possible reason is that one single copy of GFP coding gene is not enough to produce detectable amount of GFP. Considering this, we change our characterization plan to visualize bacteria through gram staining. However, since the staining of mammalian cell is not consistence, over-staining and failed destaining happening again and again, we can hardly provide any conclusive evidence to show the binding of B. hercules to tumor cell and provide significant data when comparing with the control group. In this situation, we need to come up with some other more reliable methods to detect the binding.
+
We planned to co-culture green fluorescence labelled B. hercules with HT-29 cells (colon adenocarcinoma) and HBE16 cells (Human bronchial epithelial cells) and then look for attached bacteria through their fluorescence signals. The labelled <i>B. subtilis</i> was constructed but no GFP signal could be detected. Since the GFP coding gene was integrated into the genome as a single copy, inadequate expression might had yielded undetectable signal. As an alternative, we turned to bacterial visualization using Gram stain. Yet, it appeared that not only <i>B. subtilis</i>, but also mammalian cells, could be stained by crystal violet. To make things worse, there was difficulty in achieving consistent staining in mammalian cells, since they were often over-stained or poorly destained.  Conclusive evidence, with respect to adhensions between B. hercules and tumor cells, could not be produced from the much limited method. A more reliable characterization method is thus necessary.
</li>
</li>
-
   <li>Continue verifying the tumor-apoptosis effect of BMP2 produced by B. hercules.
+
   <li><strong>Continue the verification on the tumor-apoptosis effect of BMP2 produced by B. hercules:</strong><br/>
-
Several rounds of MTT assay has been performed in order to quantify cell proliferation rate after co-culturing mammalian cell with bacteria. However, no matter how gentle we wash after co-culture in order to get rid of bacteria during MTT assay, mammalian cells will detached from the bottom of wells and lose a lot while bacteria are still in contact with mammalian cells. Therefore, other methods need to be proposed in order to characterize the effect of BMP2 to colon cancer cell.
+
Several rounds of MTT assay have been performed in order to quantify cell proliferation rates after co-culturing mammalian cell with our BMP2 secreting <i>B. subtilis</i>. The technical problem lied in the poor adhension of mammalian cells to the wells, as well as the difficulty in washing <i>B. subtilis</i> off the mammalian cells. A balanced number of washes that could retain the majority of mammalian cells inside the wells while getting rid of <i>B. subtilis</i> was not achievable. Our experience therefore called for another method to characterize the effect of BMP2 on colon cancer cells.
</li>
</li>
   </ol>
   </ol>
Line 404: Line 406:
</style>
</style>
</body>
</body>
 +
 +
<div id="Sitemap">
 +
<div id="Sitemap_Home" align="center">
 +
<p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong"><b>Home</b></a></p>
 +
</div>
 +
<div class="Sitemap_Content">
 +
<li><p><b>Team</b><p><ol>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Introduction">Introduction</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Supervisor">Supervisor</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Instructor">Instructor</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Members">Members</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Advisors">Advisors</a></p></li>
 +
</ol>
 +
</div>
 +
<div class="Sitemap_Content">
 +
<li><p><b>Project</b></p><ol>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Project_Abstraction">Abstract</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Background_and_Motive">Motive</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Overview">Design - Overview</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Module">Design - Module</a></p></li>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Target_binding">Target Binding Module</a></p>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Anti_tumor">Anti-tumor Molecule Secretion Module</a></p>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control">Regulation and Control Module</a></p>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Design_Chassis">Design - Chassis</a></p></li></ol>
 +
</div>
 +
<div class="Sitemap_Content">
 +
<li><p><b>Wet Lab</b></p><ol>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Parts_and_Device">Parts and Devices</a></p></li>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Parts_and_Device">Overview</a></p>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Construction">Construction</a></p>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Assembly">Assembly</a></p>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook">Notebook</a></p></li>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook/Logbook">Logbook</a></p>
 +
<p>-- <a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Notebook/Protocol">Protocol</a></p>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Characterization">Characterization</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Achievement">Achievement</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Future_Work">Future Work</a></p></li></ol>
 +
</div>
 +
<div class="Sitemap_Content">
 +
<li><p><b>Human Practice</b></p><ol>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Overview">Overview</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Interview">Interview</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Presentation">Presentation</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Calendar">Calendar</a></p></li></ol>
 +
</div>
 +
<div class="Sitemap_Content">
 +
<li><p><b>Extras</b></p><ol>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Medal_Requirements">Medal Requirements</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Safety">Safety</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Attribution">Attribution</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Acknowledgement">Acknowledgement</a></p></li>
 +
<li><p><a href="https://2012.igem.org/Team:HKUST-Hong_Kong/Glossary">Glossary</a></p></li></ol>
 +
</div>
 +
</div>
 +
<style type="text/css">
 +
#Sitemap{
 +
background-image:url('https://static.igem.org/mediawiki/2012/c/c0/HKUST_Campus.jpg');
 +
background-color:#ffffff;
 +
width:955px;
 +
height:auto;
 +
float:left;
 +
margin-top:5px;
 +
margin-bottom:5px;
 +
padding-bottom:5px;
 +
padding-top:5px;
 +
padding-left:4px;
 +
border:3px solid #000000;
 +
border-radius:10px;
 +
-moz-border-radius:10px;
 +
}
 +
.Sitemap_Content{
 +
background-color:#ffffff;
 +
opacity:0.8;
 +
width:173px;
 +
height:auto;
 +
float:left;
 +
margin:1px;
 +
padding-bottom:5px;
 +
padding-left:5px;
 +
border:3px solid #000000;
 +
border-radius:10px;
 +
-moz-border-radius:10px;
 +
}
 +
#Sitemap_Home{
 +
background-color:#ffffff;
 +
opacity:0.8;
 +
width:940px;
 +
height:auto;
 +
float:left;
 +
margin:1px;
 +
padding-bottom:5px;
 +
padding-left:5px;
 +
border:3px solid #000000;
 +
border-radius:10px;
 +
-moz-border-radius:10px;
 +
}
 +
</style>
</p>
</p>
Line 470: Line 569:
</div>
</div>
<!-- Served in 0.201 secs. -->
<!-- Served in 0.201 secs. -->
-
 

Latest revision as of 22:41, 26 September 2012

Team:HKUST-Hong Kong - 2012.igem.org

Future Work

Unfortunately, our wet lab only lasted for three months and we were short of time. The completeness of our project was heavily deterred. Inconclusive results from some of the modules have also increased difficulty in demonstrating the full story of B. hercules. Many characterization methods have yet to be attempted. Some experiments could be repeatd as well, and hopefully that would increase confidence in previously obtaiend data.

If more time is allowed, the following would be what we wish to pursue:

  1. Finish assembly of the parts from the three modules into pDG1661 and transformed into Bacillus subtilis:
    So far, we have inserted the BMP2 construct (BBa_K733016 and BBa_K733017) and the RPMrel construct (BBa_K733007) into the integration vector pDG1661 separately for characterization. The next step would be to piece these separated parts, as well as the remaining constructs, together to complete our B. hercules. Then we could move on to the final characterization of the strain.
  2. Continue the characterization on binding capability of B. hercules to colon adenocarcinoma HT-29:
    We planned to co-culture green fluorescence labelled B. hercules with HT-29 cells (colon adenocarcinoma) and HBE16 cells (Human bronchial epithelial cells) and then look for attached bacteria through their fluorescence signals. The labelled B. subtilis was constructed but no GFP signal could be detected. Since the GFP coding gene was integrated into the genome as a single copy, inadequate expression might had yielded undetectable signal. As an alternative, we turned to bacterial visualization using Gram stain. Yet, it appeared that not only B. subtilis, but also mammalian cells, could be stained by crystal violet. To make things worse, there was difficulty in achieving consistent staining in mammalian cells, since they were often over-stained or poorly destained. Conclusive evidence, with respect to adhensions between B. hercules and tumor cells, could not be produced from the much limited method. A more reliable characterization method is thus necessary.
  3. Continue the verification on the tumor-apoptosis effect of BMP2 produced by B. hercules:
    Several rounds of MTT assay have been performed in order to quantify cell proliferation rates after co-culturing mammalian cell with our BMP2 secreting B. subtilis. The technical problem lied in the poor adhension of mammalian cells to the wells, as well as the difficulty in washing B. subtilis off the mammalian cells. A balanced number of washes that could retain the majority of mammalian cells inside the wells while getting rid of B. subtilis was not achievable. Our experience therefore called for another method to characterize the effect of BMP2 on colon cancer cells.

Retrieved from "http://2012.igem.org/Team:HKUST-Hong_Kong/Future_Work"