Difference between revisions of "Team:AshesiGhana/Results"

 
(4 intermediate revisions by the same user not shown)
Line 97: Line 97:
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
   <center>
 
   <center>
−
     <h4><font color="#111">PPK</font></h4>
+
     <h4><font color="#111">Part Synthesis Results</font></h4>
 
     <hr class="dark">
 
     <hr class="dark">
 
   </center>
 
   </center>
  
−
<p><font color="#111">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed condimentum turpis ut aliquam pellentesque. Quisque consectetur quam lorem, ut varius elit mollis euismod. Fusce gravida orci metus, nec cursus dui pulvinar quis. Vivamus accumsan magna tortor, quis egestas magna faucibus in. Morbi enim sem, sollicitudin viverra viverra et, tempor sit amet justo. Quisque malesuada magna eu lorem egestas, quis molestie lectus sollicitudin. Donec varius magna vel eros faucibus, vel cursus eros condimentum. Donec justo lacus, tempus sed vestibulum quis, semper vitae nulla. Nam vel accumsan mi. Cras molestie, risus in hendrerit laoreet, sapien mauris ultricies enim, in fermentum ligula nibh a tellus. In vitae sollicitudin nibh. Praesent convallis consectetur vulputate.
+
<p><b>ACIDITHIOBACILLUS FEROXIDANS DESIGN PARTS</b></p>
 +
<table class="table table-bordered table-striped">
 +
<thead>
 +
        <tr>
 +
            <th>Part</th>
 +
            <th>Synthesis Status</th>
 +
           
 +
        </tr>
 +
    </thead>
 +
    <tbody>
 +
        <tr>
 +
            <td>ORIGIN OF REPLICATION <img src="https://static.igem.org/mediawiki/2017/5/52/T--AshesiGhana--orep.png"></td>           
 +
            <td>Not Synthesized Successfully</td>
 +
        </tr>
 +
        <tr>
 +
            <td>FRET-DONOR <img src="https://static.igem.org/mediawiki/2017/6/6a/T--AshesiGhana--fretdon.png" ></td>
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
        <tr>
 +
            <td>FRET-ACCEPTOR<img src="https://static.igem.org/mediawiki/2017/9/9b/T--AshesiGhana--fretacc.png"></td>
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
 
 +
    </tbody>
 +
</table>
 +
 
 +
 
 +
 
 +
<p><b>ECOLI DESIGN PARTS</b></p>
 +
<table class="table table-bordered table-striped">
 +
<thead>
 +
        <tr>
 +
            <th>Part</th>
 +
            <th>Synthesis Status</th>
 +
           
 +
        </tr>
 +
    </thead>
 +
    <tbody>
 +
 +
        <tr>
 +
            <td>FRET-DONOR <img src="https://static.igem.org/mediawiki/2017/6/6a/T--AshesiGhana--fretdon.png" ></td>
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
        <tr>
 +
            <td>FRET-ACCEPTOR<img src="https://static.igem.org/mediawiki/2017/9/9b/T--AshesiGhana--fretacc.png"></td>
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
 
 +
              <tr>
 +
            <td>HIGH POTENTIAL IRON SULFUR PROTEIN (HIPIP)<img src="https://static.igem.org/mediawiki/2017/7/75/T--AshesiGhana--hipip.png"></td>           
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
        <tr>
 +
            <td>PH RESISTANCE<img src="https://static.igem.org/mediawiki/2017/8/8e/T--AshesiGhana--hpresist.png"></td>
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
 
 +
              <tr>
 +
            <td>TETRATHIONATE HYDROLASE<img src="https://static.igem.org/mediawiki/2017/3/36/T--AshesiGhana--tetrahydro.png"></td>           
 +
            <td>Synthesized Successfully</td>
 +
        </tr>
 +
 
 +
            <tr>
 +
            <td>MRUBY 2<img src=""></td>           
 +
            <td>Not Synthesized Successfully</td>
 +
        </tr>
 +
 
 +
            <tr>
 +
            <td>NowGFP<img src=""></td>           
 +
            <td>Not Synthesized Successfully</td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
 
 +
 
  
−
Vestibulum faucibus augue a pretium interdum. Phasellus eros felis, tincidunt eget odio a, imperdiet consequat ipsum. Duis bibendum mattis efficitur. Etiam auctor id elit at fringilla. Donec pretium, ex id pellentesque varius, risus lacus pellentesque tortor, non tincidunt eros libero vel mi. Morbi viverra tempor mattis. Curabitur mattis sed dolor in aliquet. Maecenas nec metus at nibh gravida commodo id vel dolor.
 
  
−
Morbi commodo cursus lacus, et ullamcorper nibh lacinia in. Mauris lectus enim, sodales vel dolor eget, volutpat volutpat purus. Quisque vitae odio pellentesque, pulvinar lorem eu, congue ligula. Morbi magna sem, pharetra sed sagittis quis, aliquam sit amet purus. Aliquam eget est ut ligula facilisis gravida ut ut ante. Nullam quis venenatis nulla. Nam facilisis ex in ligula consequat posuere. Pellentesque id arcu mattis, gravida elit a, hendrerit ante. Integer mollis justo sed vestibulum vehicula.</font></p>
 
 
   </div>
 
   </div>
  
Line 111: Line 183:
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
   <center>
 
   <center>
−
     <h4><font color="#111">Eut Bacterial Microcompartment Expression</font></h4>
+
     <h4><font color="#111">PARTS DIGESTED AND TRANSFORMED</font></h4>
 
     <hr class="dark">
 
     <hr class="dark">
 
   </center>
 
   </center>
  
−
<p><font color="#111">Previous iGem teams have found that microcompartments have proved difficult to work with. Because of this, we thought it would be beneficial to work on optimising the formation of micro-compartments. These three constructs (EutS, EutMN and EutLK) were each combined with an independent inducible promoter, to enable variable synthesis of micro-compartment proteins and allow us to optimise micro-compartment formation with varying induction levels. We designed a collection of experiments, varying in complexity in order to prove that microcompartment formation was induced by our promoters. We also wanted to understand if microcompartment protein synthesis induced stress within our chassis and affected growth.
+
<p><font color="#111"><br>
 +
 
 +
<ol style="font-size:16px;">
 +
 
 +
<li><b>Make up ligation reaction as below</b><br></li>
 +
<li><b>High Potential Iron Sulphur Protein (HiPiP)</b><br></li>
 +
<li><b>Tetrathionate  Hydrolase</b><br></li>
 +
<li><b>Fret-Donor </b><br></li>
 +
<li><b>Fret-Acceptor</b><br></li>
 +
</ol>
 
<br>
 
<br>
 
<br>
 
<br>
−
We induced our constructs with their respective reagents for 4 hours and 20 hours before collecting soluble and insoluble proteins. These samples were then run on a 12% Tris-Glycine SDS-Page gel. Unfortunately, we were unable to see any bands of increased intensity, see figure 1. and the corresponding table 1. for the bands we were expecting.   
+
<b>Complete Total Number of Transformations = 5</b><br>
 +
<b>Successful number of Transformations = 2</b><br>
 +
<b>Number of failed Transformations = 3</b><br>
 
</font></p>
 
</font></p>
 
   </div>
 
   </div>
 
<br>
 
<br>
 
<br>
 
<br>
−
<img src="https://static.igem.org/mediawiki/2017/2/25/Predicted_protein_sizes_sds_page.png" width="800" height="317">
+
 
  
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
   <center>
 
   <center>
−
     <h4><font color="#111">Localization Tag</font></h4>
+
     <h4><font color="#111">Results pH Resistance Gene</font></h4>
 
     <hr class="dark">
 
     <hr class="dark">
 
   </center>
 
   </center>
−
<p><font color="#111">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed condimentum turpis ut aliquam pellentesque. Quisque consectetur quam lorem, ut varius elit mollis euismod. Fusce gravida orci metus, nec cursus dui pulvinar quis. Vivamus accumsan magna tortor, quis egestas magna faucibus in. Morbi enim sem, sollicitudin viverra viverra et, tempor sit amet justo. Quisque malesuada magna eu lorem egestas, quis molestie lectus sollicitudin. Donec varius magna vel eros faucibus, vel cursus eros condimentum. Donec justo lacus, tempus sed vestibulum quis, semper vitae nulla. Nam vel accumsan mi. Cras molestie, risus in hendrerit laoreet, sapien mauris ultricies enim, in fermentum ligula nibh a tellus. In vitae sollicitudin nibh. Praesent convallis consectetur vulputate.
 
  
−
Vestibulum faucibus augue a pretium interdum. Phasellus eros felis, tincidunt eget odio a, imperdiet consequat ipsum. Duis bibendum mattis efficitur. Etiam auctor id elit at fringilla. Donec pretium, ex id pellentesque varius, risus lacus pellentesque tortor, non tincidunt eros libero vel mi. Morbi viverra tempor mattis. Curabitur mattis sed dolor in aliquet. Maecenas nec metus at nibh gravida commodo id vel dolor.
+
<p><font color="#111"><b>Total number of Transformations = 3</b><br>
 +
<b>Successful number of Transformations = 2</b><br>
 +
<b>Number of failed transformations = 1</b><br>
 +
</font></p>
  
−
Morbi commodo cursus lacus, et ullamcorper nibh lacinia in. Mauris lectus enim, sodales vel dolor eget, volutpat volutpat purus. Quisque vitae odio pellentesque, pulvinar lorem eu, congue ligula. Morbi magna sem, pharetra sed sagittis quis, aliquam sit amet purus. Aliquam eget est ut ligula facilisis gravida ut ut ante. Nullam quis venenatis nulla. Nam facilisis ex in ligula consequat posuere. Pellentesque id arcu mattis, gravida elit a, hendrerit ante. Integer mollis justo sed vestibulum vehicula.</font></p>
+
<p><font color="#111"><b>Total number of gels runs = 3</b><br>
−
  </div>
+
<b>Successful number of gel runs = 1</b><br>
 +
<b>Number of failed gel runs = 2</b><br>
 +
</font></p>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2017/f/f8/T--AshesiGhana--gelimage.jpg">
 +
<br>
 +
<p><font color="#111">From the gel image in figure 1 above it is evident that the pH resistance gene was inserted in the plasmid, however due to a small concentration of the DNA present, the band of the insert seemed to be blurred out.
 +
</font></p>
 +
 
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2017/a/aa/T--AshesiGhana--effgraph.jpg">
 +
<br>
 +
<p><font color="#111">From the results obtained and displayed in the graph above, it is evident that the pH resistance gene is effective for pH levels ranging from 5.5 to 7.5, where 7.5 is the pH level of the original media for the bacterial growth. It is also observed that, the resistance gene was not effective with pH levels 4.5 and below, resulting in no bacterial growth over time.
 +
</font></p>
 +
</div>
  
  
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
<div class="clear" style="padding: 0px 12vw; border: 3px solid transparent; background-color: #fbf9f8!important">
 
   <center>
 
   <center>
−
     <h4><font color="#111">Localization Tag Characterization using Microscopy</font></h4>
+
     <h4><font color="#111">Results FRET Donor</font></h4>
 
     <hr class="dark">
 
     <hr class="dark">
 
   </center>
 
   </center>
  
−
<p><font color="#111">We aimed to confirm our constructs were working via separate means so we designed all of the tag-promoter constructs with mCherry attached so that they could be visualised via fluorescent microscopy. We visualised the tag-promoter constructs using mCherry to check the distribution of the tag throughout the cell, whether the tag localised in the presence of Eut and to see the level of expression based on fluorescence level.<br>
+
<p><font color="#111"><b>Total number of Transformations = 5</b><br>
 +
<b>Successful number of Transformations = 2</b><br>
 +
<b>Number of failed transformations = 3</b><br>
 +
</font></p>
 +
 
 +
<p><font color="#111"><b>Total number of gels runs = 3</b><br>
 +
<b>Successful number of gel runs = 0</b><br>
 +
<b>Number of failed gel runs = 3</b><br>
 +
</font></p>
 
<br>
 
<br>
−
<center>
+
<img src="https://static.igem.org/mediawiki/2017/d/d9/T--AshesiGhana--fretdoneff.png">
−
<img src="https://static.igem.org/mediawiki/2017/2/2d/PromoterComparison.png" width="800" height="317">
+
 
−
</center>
+
 
<br>
 
<br>
−
<p><font color="#111">As expected, without the expression of any Eut subunits, the tag showed a homogeneous distribution throughout the cells with no localization. The difference in fluorescence was most pronounced between low promoter and the other two as shown (in another figure? There’s a graph showing medium and high expression levels as very similar).<br>
 
 
<br>
 
<br>
−
As medium promoter and high promoter had a similar level of expression, only low and high were combined with Eut subunits for visualization.<br>
 
 
<br>
 
<br>
−
Levels of fluorescence from Low+EutS were too low to properly visualize. High+EutS showed slightly heterogeneous distribution of fluorescence, the fluorescence was slightly granular with some brighter areas and some darker areas but no well-defined localization.<br>
 
 
<br>
 
<br>
−
Low+EutSMN images were very dim but visualization was possible. The fluorescence was granular but mainly heterogeneous throughout the cell. High+EutSMN showed quite well defined localization in a number of cells.<br>
+
<img src="https://static.igem.org/mediawiki/2017/c/c2/T--AshesiGhana--abso.png">
−
<br>
+
−
SMNLK was visualized with the low promoter as successful construction of High+EutSMNLK was not possible before we ran out of time. Fluorescence from Low+EutSMNLK showed quite well-defined localization with a number of cells showing relatively round accumulations of fluorescent tag, suggesting proper BMC formation.<br>
+
−
<br>
+
−
It is important to note that the majority of the accumulations seen occur at or near the end of the cells. This could be indicative of protein aggregates and not proper BMC formation.<br>
+
−
<br>
+
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2017/4/47/LowPromoterComparison2.png" width="800" height="317">
+
−
</center>
+
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2017/8/81/HighPromoterComparison2.png" width="800" height="317">
+
−
</center>
+
−
<br>
+
−
<p><font color="#111">After successful expression of Low+EutSMNLK we decided to transform EutLK-Low-PduD-mCherry-PPK and EutSMN together. We reasoned that this SMNLK+PPK construct would allow us to determine whether it actually worked via DAPI staining. The polyphosphate chains produced by PPK can be stained using DAPI and as such both mCherry and DAPI could be visualised in the same cells. This would allow us to see any co-localisation of the two signals, demonstrating successful Eut subunit expression, successful tag localisation and successful PPK activity.<br>
+
−
<br>
+
−
A control DAPI stain was performed along side Medium promoter tag expression to inspect the DAPI distribution in the absence of polyphosphate and whether the staining procedure interfered with mCherry distribution.<br>
+
−
<br>
+
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2017/a/a9/MidDAPIcontrol.png" width="800" height="317">
+
−
</center>
+
−
<br>
+
−
<p><font color="#111">As shown, the distribution of both mCherry and DAPI were homogeneous with no obvious clumping or accumulation. This suggested that if our construct was working properly, we would see an accumulation of fluorescent signal for both mCherry and DAPI in the same place within the cell.<br>
+
−
<br>
+
−
So in line with this, we DAPI stained a 24h induction of Low+EutSMNLK+PPK:<br>
+
−
<br>
+
−
<center>
+
−
<img src="https://static.igem.org/mediawiki/2017/d/d0/SMNLKDAPI1.png" width="300" height="300">
+
−
<img src="https://static.igem.org/mediawiki/2017/b/b5/SMNLKDAPI2.png" width="300" height="300">
+
−
<img src="https://static.igem.org/mediawiki/2017/f/f6/SMNLKDAPI4.png" width="300" height="300">
+
−
</center>
+
−
<br>
+
−
<p><font color="#111">As shown, there is a heterogeneous distribution of fluorescence within the cells for both signals and they are approximately in the same areas. This heterogeneous distribution of DAPI indicates the presence of polyphosphate and proves the activity of our PPK along with its successful localization into our BMC. The localisation can be determined using the physical location of both fluorescence signals within the cell.<br>
+
 
<br>
 
<br>
 +
<p><font color="#111">From figure 4 and 3 above, it is observed that the absorption and fluorescence in the sample increased over time as the bacteria growth increased, however the change was not significant and this concludes that the FRET-Donor is not as effective as expected. This could however be attributed to the fact that concentration of the Donor DNA isn’t enough to produce the expected high absorption and fluorescence values.
 
</font></p>
 
</font></p>
 +
</div>
  
−
 
−
</div>
 
  
 
</body>
 
</body>
 
</html>
 
</html>
 
{{Template:AshesiGhana/Footer}}
 
{{Template:AshesiGhana/Footer}}

Latest revision as of 03:01, 2 November 2017

Ashesi University
College
HOME
TEAM
PROJECT
Overview
Description
Results
Demonstration
Medal Criteria
Achievement
NOTEBOOK
Protocols
Diary
Lab Book
InterLab
Measurement
PARTS
Parts
Basic Parts
Composite Parts
Improved Parts
Part Collection
HUMAN PRACTICES
Silver HP
Water Industry
Integrated and Gold
Intellectual Property
Public Engagement
Entrepreneurship
MODELLING
Overview
D. O. Experiment
Continuous Culture
PHO Operon
JUDGING FORM

Results


Part Synthesis Results

ACIDITHIOBACILLUS FEROXIDANS DESIGN PARTS

Part Synthesis Status
ORIGIN OF REPLICATION Not Synthesized Successfully
FRET-DONOR Synthesized Successfully
FRET-ACCEPTOR Synthesized Successfully

ECOLI DESIGN PARTS

Part Synthesis Status
FRET-DONOR Synthesized Successfully
FRET-ACCEPTOR Synthesized Successfully
HIGH POTENTIAL IRON SULFUR PROTEIN (HIPIP) Synthesized Successfully
PH RESISTANCE Synthesized Successfully
TETRATHIONATE HYDROLASE Synthesized Successfully
MRUBY 2 Not Synthesized Successfully
NowGFP Not Synthesized Successfully

PARTS DIGESTED AND TRANSFORMED


  1. Make up ligation reaction as below
  2. High Potential Iron Sulphur Protein (HiPiP)
  3. Tetrathionate Hydrolase
  4. Fret-Donor
  5. Fret-Acceptor


Complete Total Number of Transformations = 5
Successful number of Transformations = 2
Number of failed Transformations = 3



Results pH Resistance Gene

Total number of Transformations = 3
Successful number of Transformations = 2
Number of failed transformations = 1

Total number of gels runs = 3
Successful number of gel runs = 1
Number of failed gel runs = 2



From the gel image in figure 1 above it is evident that the pH resistance gene was inserted in the plasmid, however due to a small concentration of the DNA present, the band of the insert seemed to be blurred out.



From the results obtained and displayed in the graph above, it is evident that the pH resistance gene is effective for pH levels ranging from 5.5 to 7.5, where 7.5 is the pH level of the original media for the bacterial growth. It is also observed that, the resistance gene was not effective with pH levels 4.5 and below, resulting in no bacterial growth over time.

Results FRET Donor

Total number of Transformations = 5
Successful number of Transformations = 2
Number of failed transformations = 3

Total number of gels runs = 3
Successful number of gel runs = 0
Number of failed gel runs = 3







From figure 4 and 3 above, it is observed that the absorption and fluorescence in the sample increased over time as the bacteria growth increased, however the change was not significant and this concludes that the FRET-Donor is not as effective as expected. This could however be attributed to the fact that concentration of the Donor DNA isn’t enough to produce the expected high absorption and fluorescence values.