Difference between revisions of "Team:Hong Kong UCCKE/Experiments"

 
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<h3 class="sectiontitle">Assay</h3>
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<p style="text-align:left !important;">We have done 4 assays, which are Assay A, Assay B, Assay C and Assay D on the cultured cells to test whether part BBa_K2197300, BBa_K2197400, BBa_K2197500 and BBa_K2197502 are valid. Protocols are documented on the protocol page. The design and results will be discussed in detail below.</p>
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<h3 class="sectiontitle">Assay A</h3>
 
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                <p style="text-align:left !important;">We have done assays on the cultured cells.</p>
 
  
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<p>The aim of doing these assays was to test whether part 300,400 and 500 are valid. We expect there will be a positively proportional trend when putting part 300 into increasing concentrations of uric acid. The higher the concentration is, the more GFP expression.  
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<p><font size="4">Design</font></p>
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Below I explain the steps and calculations involved in these assays in detail.</p>
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<p style="text-align:left !important;"> Assay A is an experiment designed to prove the validity of BBa_K2197300. By incubating engineered cells with different concentrations of uric acid and measuring the green fluorescence level in a plate reader, it is expected a positively proportional trend be shown. As theoretically, the higher the concentration is, the more the GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration.</p>
  
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<p>We first centrifuge the cell and resuspend it with PSD. We then add uric acid into the cell for growth. We also grow cell with uric acid in different concentrations for the second experiment. We expect that there will be an increase in GFP expression when it was put in higher uric acid concentration. When there is no uric acid, or in very low concentration, the strong repressor KRAB-HucR will stop the expression and thus no GFP expression. For Column 1-3, 4-6, 7-9, and 10-12, they are uric acid with water, K2197300, K2197400 with K2197300, and K2197500 with K2197300 respectively.</p>
 
  
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<p style="text-align:left !important;">General Steps:</p>
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<ol start="1"><li>Centrifuge 4 ml of cells and discard the LB</li>
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<p><font size="4">Steps and Result</font></p>
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<li>Resuspend them with 200ul of PBS</li>
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<p style="text-align:left !important;">We first pipette the cells into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we put it into the plate reader to measure the green fluorescence level. </p><br>
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<li>Pipette 50ul of water into A4-A12</li>
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<li>Pipette 50 ul of 1x10^-4 mg/dL uric acid into B4-B12</li>
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<p style="text-align:left !important;">The result is shown as below (Click me for raw data). </p>
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<li>Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:</li>
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<div class="pop">
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<div class="table-responsive">
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<a href="https://static.igem.org/mediawiki/2017/f/fc/T--Hong_Kong_UCCKE--300graph.jpg" title="Adding uric acid to the cell">
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<table><tr><th></th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>Concentration of Uric Acid</th></tr><tr><td>A</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0</td></tr><tr><td>B</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1x10^-4</td></tr><tr><td>C</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>2x10^-4</td></tr><tr><td>D</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4x10^-4</td></tr><tr><td>E</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>6x10^-4</td></tr><tr><td>F</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>8x10^-4</td></tr><tr><td>G</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1x10^-3</td></tr><tr><td>H</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1x10^-3</td></tr></table>
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<img src="https://static.igem.org/mediawiki/2017/f/fc/T--Hong_Kong_UCCKE--300graph.jpg" alt="Adding uric acid to the cell"style="width:100%;">
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</a>
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</div><br>
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<p style="text-align:left !important;">There is no significant trend shown. When higher concentrations of uric acid are added, the levels of green fluorescence measured are similar to that in lower concentrations. Therefore, we carry out another experiment, Assay B, after receiving the advice from 2017 team Hong Kong-CUHK.
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</p><br>
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<div class="col-md-4">
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<div style="text-align:left;">
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<a href="https://static.igem.org/mediawiki/2017/3/3b/T--Hong_Kong_UCCKE--uricacid.jpg" title="Assay A"><img src="https://static.igem.org/mediawiki/2017/3/3b/T--Hong_Kong_UCCKE--uricacid.jpg" style="height:auto; width:100%; max-width:500px; display: block;" alt="Assay A"></a><span class="imgcaption">Assay A</span>
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<a href="https://static.igem.org/mediawiki/2017/e/e0/T--Hong_Kong_UCCKE--96wellplate.jpg" title="Assay A"><img src="https://static.igem.org/mediawiki/2017/e/e0/T--Hong_Kong_UCCKE--96wellplate.jpg" style="height:auto; width:100%; max-width:500px; display: block;" alt="Assay A"></a><span class="imgcaption">Assay A</span>
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</div>
 
</div>
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<li>Add 50 ul of cultured Cells (Competent cells into Column 1-3; K2197300 into Column 4-6; K2197400 into Column 7-9 and K2197500 into Column 10-12) and let it set for 1 hour</li>
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<li>Add 50 ul of cultured Cells ( K2197300) into Column 7-12</li>
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<li>Let it set for 1-2 hours</li>
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<li>Place it in the plate reader to measure the Green Fluorescein </li></ol>
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<h3 class="sectiontitle" style="clear:both;">Results</h3>
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<h3 class="sectiontitle">Assay B</h3>
 
                         <div class="divider"></div>
 
                         <div class="divider"></div>
  
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<p><font size="4">Design</font></p>
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<p style="text-align:left !important;"> Assay B is another experiment designed to prove the validity of BBa_K2197300. By growing engineered cells with different concentrations of uric acid and measuring the green fluorescence level in a plate reader, we expect a positively proportional trend will be shown. As theoretically, the higher the concentration is, the more the GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration.</p>
 +
 +
<p><font size="4">Steps and Result</font></p>
 +
<p style="text-align:left !important;">We first grew cells in LB broth overnight. Then we cultured cells from the same tube with different concentrations of uric acid-LB mixed. We then incubate the cells for 1 hour and then pipette them into the 96-well plate. After that, we put it into the plate reader to measure the green fluorescence level. </p><br>
 +
 +
<p style="text-align:left !important;">The result is shown as below (Click to see raw data). </p>
 
<div class="pop">
 
<div class="pop">
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<a href="https://static.igem.org/mediawiki/2017/6/6e/T--Hong_Kong_UCCKE--experiment2.png">
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<a href="https://static.igem.org/mediawiki/2017/2/21/T--Hong_Kong_UCCKE--300wifcellgraph.jpg" title="Growing the cell with uric acid">
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<img src="https://static.igem.org/mediawiki/2017/6/6e/T--Hong_Kong_UCCKE--experiment2.png" alt="" style="width:100%;">
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<img src="https://static.igem.org/mediawiki/2017/2/21/T--Hong_Kong_UCCKE--300wifcellgraph.jpg" alt="Growing the cell with uric acid"style="width:100%;">
 
</a>
 
</a>
 
</div>
 
</div>
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<p style="text-align:left !important;">There is also no significant trend shown. When higher concentrations of uric acid are added, the amount of GFP measured is similar to that in lower concentration. Therefore, we tried to figure out the problem. After double checking the sequence, we found out that the DNA sequence we ordered from IDT is different from what we designed, in which the sequence 'tactagag' is missing. GFP E0040, Terminator B0010, K2197302 and K2197303 are affected.  Referring to the mechanism of the promoter, if HucO is missing, the repressor protein mUTS can not bind to it. Thus, the amount of GFP expression cannot be restricted and no trend can be shown. Also, the GFP is seriously affected, therefore, GFP cannot be expressed.
 +
</p><br>
  
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                <p style="text-align:left !important;">The result is shown as above, which there is no significant trend when higher concentrations of uric acid is added. After double checking the sequence, we found out that the DNA we ordered is different from  what we designed, which the operating site HucO is missing. Therefore, mUTS cannot bind to it, resulting a negative feedback in terms of GFP expression. (project design: https://2017.igem.org/Team:Hong_Kong_UCCKE/Design)
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</div>
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However, it is possible that the GFP itself is not working or the repressor KRAB-HucR is not strong enough. More experiments to be done to figure out the answer. </p>
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<div class="col-md-4 pop">
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<h3 class="sectiontitle" style="clear:both;">Calibration</h3>
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<div style="text-align:left;">
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<a href="https://static.igem.org/mediawiki/2017/9/91/T--Hong_Kong_UCCKE--cellsgrowinuricacid-LBmixed.jpg" title="Assay B"><img src="https://static.igem.org/mediawiki/2017/9/91/T--Hong_Kong_UCCKE--cellsgrowinuricacid-LBmixed.jpg" style="height:auto; width:100%; max-width:500px; display: block;" alt="Assay B"></a><span class="imgcaption">Assay B</span>
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</div>
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<div class="col-md-8">
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<h3 class="sectiontitle">Assay C</h3>
 
                         <div class="divider"></div>
 
                         <div class="divider"></div>
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                <p style="text-align:left !important;">We expect that there will be a proportional trend when putting K2197300 with uric acid. When we have a positive result in assays, we can use it to determine the effectiveness of K2197400 and K2197500. When uric acid presents, K2197400 can turn uric acid into allantoin by smUOX and K2197500 can absorb uric acid by YgfU. With the result of K2197300, we can estimate the amount of reduced uric acid by comparing the light intensity measured.
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</p>
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<p><font size="4">Design</font></p>
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<p>Although we do not have a positive result for K2197300, we planned to do the following experiments to interpret the effectiveness of K2197400. For K2197400, we will first add 50ul of uric acid, competent cell and K2197400 to column 1 and 50ul of uric acid, K2197400 and K2197300 to column 2. By comparing the result, we will know the efficiency of K2197400 in turning uric acid into allantoin.</p>
+
<p style="text-align:left !important;"> Assay C is an experiment designed to prove the validity of BBa_K2197400. By incubating engineered cells (BBa_K2197400) and Competent cells with different concentrations of uric acid, then adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence level in a plate reader. We expect that the columns with engineered cells (BBa_K2197400) will have less green fluorescence expressed when comparing to the column containing competent cells. As theoretically, the higher the concentration is, the more the smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which expresses more GFP when there is more uric acid, the green fluorescence level of the columns with engineered cells(BBa_K2197400) will be lower than the columns with competent cells although they were in the same concentration at the beginning</p>
 +
 
 +
<p><font size="4">Steps and Result</font></p>
 +
<p style="text-align:left !important;">We first pipette the cells (BBa_2197400, competent cells) into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we add in engineered cells (BBa_K2197300) into the wells and incubate them for another 30 minutes. Then, we put it into the plate reader for the measurement of green fluorescence level. </p><br>
 +
<p style="text-align:left !important;">However, due to the problem of BBa_K2197300, in which there is no difference of the green fluorescence levels between the one with lower uric acid concentration and higher uric acid concentration, we cannot determine whether it can really lower the level of uric acid.</p><br>
 +
 
 +
 
 +
<h3 class="sectiontitle">Assay D</h3>
 +
                        <div class="divider"></div>
 +
 
 +
<p><font size="4">Design</font></p>
 +
<p style="text-align:left !important;"> Assay D is an experiment designed to prove the validity of BBa_K2197500 and BBa_K2197502. By incubating engineered cells (BBa_K2197500), cells (BBa_K2197502) and Competent cells with different concentrations of uric acid, then adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence level in a plate reader. We expect that the columns with engineered cells (BBa_K2197500) and engineered cells (BBa_K2197502) will have less green fluorescence expressed when comparing to the column containing competent cells. As theoretically, engineered cells (BBa_K2197500) will absorb uric acid into the cell and for engineered cells (BBa_K2197502), the higher the concentration is, the more uric acid will be absorbed into the cells. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence level of the columns with engineered cells (BBa_K2197500 and BBa_K2197502) will be lower than the columns with competent cells although they were in the same concentration at the beginning.</p>
 +
 
 +
<p><font size="4">Steps and Result</font></p>
 +
<p style="text-align:left !important;">We first pipette the cells (BBa_2197500, BBa_K2197502, competent cells) into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we add engineered cells (BBa_K2197300) into the wells and incubate for another 30 minutes. Then, we put it into the plate reader for the measurement of green fluorescence level. </p><br>
 +
<p style="text-align:left !important;">However, due to the problem of BBa_K2197300, in which there is no difference of the green fluorescence levels between the one with lower uric acid concentration and higher uric acid concentration, we cannot determine whether it can really lower the level of uric acid.</p>
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Latest revision as of 13:10, 21 November 2017

Experiments

We have done 4 assays, which are Assay A, Assay B, Assay C and Assay D on the cultured cells to test whether part BBa_K2197300, BBa_K2197400, BBa_K2197500 and BBa_K2197502 are valid. Protocols are documented on the protocol page. The design and results will be discussed in detail below.

Assay A

Design

Assay A is an experiment designed to prove the validity of BBa_K2197300. By incubating engineered cells with different concentrations of uric acid and measuring the green fluorescence level in a plate reader, it is expected a positively proportional trend be shown. As theoretically, the higher the concentration is, the more the GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration.

Steps and Result

We first pipette the cells into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we put it into the plate reader to measure the green fluorescence level.


The result is shown as below (Click me for raw data).

Adding uric acid to the cell

There is no significant trend shown. When higher concentrations of uric acid are added, the levels of green fluorescence measured are similar to that in lower concentrations. Therefore, we carry out another experiment, Assay B, after receiving the advice from 2017 team Hong Kong-CUHK.


Assay AAssay A Assay AAssay A

Assay B

Design

Assay B is another experiment designed to prove the validity of BBa_K2197300. By growing engineered cells with different concentrations of uric acid and measuring the green fluorescence level in a plate reader, we expect a positively proportional trend will be shown. As theoretically, the higher the concentration is, the more the GFP will be expressed. It is because the strong repressor KRAB-HucR will stop the expression of GFP when there is no uric acid, or in very low concentration.

Steps and Result

We first grew cells in LB broth overnight. Then we cultured cells from the same tube with different concentrations of uric acid-LB mixed. We then incubate the cells for 1 hour and then pipette them into the 96-well plate. After that, we put it into the plate reader to measure the green fluorescence level.


The result is shown as below (Click to see raw data).

Growing the cell with uric acid

There is also no significant trend shown. When higher concentrations of uric acid are added, the amount of GFP measured is similar to that in lower concentration. Therefore, we tried to figure out the problem. After double checking the sequence, we found out that the DNA sequence we ordered from IDT is different from what we designed, in which the sequence 'tactagag' is missing. GFP E0040, Terminator B0010, K2197302 and K2197303 are affected. Referring to the mechanism of the promoter, if HucO is missing, the repressor protein mUTS can not bind to it. Thus, the amount of GFP expression cannot be restricted and no trend can be shown. Also, the GFP is seriously affected, therefore, GFP cannot be expressed.


Assay BAssay B

Assay C

Design

Assay C is an experiment designed to prove the validity of BBa_K2197400. By incubating engineered cells (BBa_K2197400) and Competent cells with different concentrations of uric acid, then adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence level in a plate reader. We expect that the columns with engineered cells (BBa_K2197400) will have less green fluorescence expressed when comparing to the column containing competent cells. As theoretically, the higher the concentration is, the more the smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which expresses more GFP when there is more uric acid, the green fluorescence level of the columns with engineered cells(BBa_K2197400) will be lower than the columns with competent cells although they were in the same concentration at the beginning

Steps and Result

We first pipette the cells (BBa_2197400, competent cells) into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we add in engineered cells (BBa_K2197300) into the wells and incubate them for another 30 minutes. Then, we put it into the plate reader for the measurement of green fluorescence level.


However, due to the problem of BBa_K2197300, in which there is no difference of the green fluorescence levels between the one with lower uric acid concentration and higher uric acid concentration, we cannot determine whether it can really lower the level of uric acid.


Assay D

Design

Assay D is an experiment designed to prove the validity of BBa_K2197500 and BBa_K2197502. By incubating engineered cells (BBa_K2197500), cells (BBa_K2197502) and Competent cells with different concentrations of uric acid, then adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence level in a plate reader. We expect that the columns with engineered cells (BBa_K2197500) and engineered cells (BBa_K2197502) will have less green fluorescence expressed when comparing to the column containing competent cells. As theoretically, engineered cells (BBa_K2197500) will absorb uric acid into the cell and for engineered cells (BBa_K2197502), the higher the concentration is, the more uric acid will be absorbed into the cells. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence level of the columns with engineered cells (BBa_K2197500 and BBa_K2197502) will be lower than the columns with competent cells although they were in the same concentration at the beginning.

Steps and Result

We first pipette the cells (BBa_2197500, BBa_K2197502, competent cells) into different concentrations of uric acid and incubate them in 96 well plate for 30 minutes. After that, we add engineered cells (BBa_K2197300) into the wells and incubate for another 30 minutes. Then, we put it into the plate reader for the measurement of green fluorescence level.


However, due to the problem of BBa_K2197300, in which there is no difference of the green fluorescence levels between the one with lower uric acid concentration and higher uric acid concentration, we cannot determine whether it can really lower the level of uric acid.

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