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

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<p><font size="4">Design</font></p>
 
<p><font size="4">Design</font></p>
 
<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 concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more 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 style="text-align:left !important;"> Assay B is another experiment designed to prove the validity of BBa_K2197300. By growing engineered cells with different concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more 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><font size="4">Steps and Result</font></p>
 
<p><font size="4">Steps and Result</font></p>
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<p><font size="4">Design</font></p>
 
<p><font size="4">Design</font></p>
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<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 concentration of uric acid, and adding engineered cells (BBa_K2197300) after 1.5 hours , we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197400) will have less green fluorescence while comparing it to the column containing competent cells. As theoretically, the higher the concentration is, the more smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence 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>
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<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 concentration of uric acid, and adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197400) will have less green fluorescence while comparing it to the column containing competent cells. As theoretically, the higher the concentration is, the more smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence 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>
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<p><font size="4">Steps and Result</font></p>
 
<p><font size="4">Steps and Result</font></p>
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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 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. </p><br>
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<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 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. </p><br>
  
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<p style="text-align:left !important;">However, due to the problem of missing the operating site in K2197300, which there is no difference of the green fluorescence 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>
+
<p style="text-align:left !important;">However, due to the problem of missing the operating site in K2197300, which there is no difference of the green fluorescence 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>
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<div class="pop">
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<h3 class="sectiontitle">Assay D</h3>
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                        <div class="divider"></div>
  
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<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 concentration of uric acid, and adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197500) and engineered cells(BBa_K2197502) will have less green fluorescence while comparing it 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 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>
  
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<p style="text-align:left !important;">General Steps:</p>
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<p><font size="4">Steps and Result</font></p>
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<ol start="1"><li>Centrifuge 4 ml of cells and discard the LB</li>
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<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. </p><br>
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<li>Resuspend them with 200ul of PBS</li>
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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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<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="table-responsive">
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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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</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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<p style="text-align:left !important;">However, due to the problem of missing the operating site in K2197300, which there is no difference of the green fluorescence 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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                        <div class="divider"></div>
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<div class="pop">
 
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<a href="https://static.igem.org/mediawiki/2017/6/65/T--Hong_Kong_UCCKE--growwithuricacid.png" title="Growing the cell with uric acid">
 
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<img src="https://static.igem.org/mediawiki/2017/6/65/T--Hong_Kong_UCCKE--growwithuricacid.png" alt="Growing the cell with uric acid"style="width:100%;">
 
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</a>
 
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</div>
 
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<P></P>
 
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<P></P>
 
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<div class="pop">
 
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<a href="https://static.igem.org/mediawiki/2017/4/4c/T--Hong_Kong_UCCKE--adduricacid.png" title="Adding uric acid to the cell">
 
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<img src="https://static.igem.org/mediawiki/2017/4/4c/T--Hong_Kong_UCCKE--adduricacid.png" alt="Adding uric acid to the cell"style="width:100%;">
 
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</a>
 
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</div>
 
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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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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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<h3 class="sectiontitle" style="clear:both;">Calibration</h3>
 
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                        <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>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>
 
  
 
</div>
 
</div>

Revision as of 13:38, 31 October 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 included in the protocol page and we will talk about the design and the result below.

Assay A

Design

Assay A is the experiment designed to prove the validity of BBa_K2197300. By incubating engineered cells with different concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more 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 for the measurement of green fluorescence.


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

Adding uric acid to the cell

There is no significant trend shown. When higher concentrations of uric acid is added the amount of GFP measured is similar to the lower concentration ones. Therefore, we carry out another experiment, Assay B, after receiving the advice from Hong_Kong_CUHK Team.


Assay B

Design

Assay B is another experiment designed to prove the validity of BBa_K2197300. By growing engineered cells with different concentration of uric acid and measure the Green fluorescence in a plate reader, we expect that there will be a positively proportional trend as theoretically, the higher the concentration is, the more 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 grow 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 for the measurement of green fluorescence.


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 is added the amount of GFP measured is similar to the lower concentration ones. Therefore, we tried to figure out the problem. After double checking the sequence, we found out that the DNA we ordered from IDT is different from what we designed, which the operating site HucO is missing. 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. However, it is possible that the GFP itself is not working or the repressor KRAB-HucR is not strong enough to repress it. And more experiments should be done to figure out the answer.


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 concentration of uric acid, and adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197400) will have less green fluorescence while comparing it to the column containing competent cells. As theoretically, the higher the concentration is, the more smUOX will be expressed and this will catalyse uric acid into allantoin. So by adding engineered cells (BBa_K2197300) which express more GFP when there is more uric acid, the green fluorescence 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 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.


However, due to the problem of missing the operating site in K2197300, which there is no difference of the green fluorescence 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 concentration of uric acid, and adding engineered cells (BBa_K2197300) after 30 minutes, we measure the Green fluorescence in a plate reader. We expect that the columns with engineered cells(BBa_K2197500) and engineered cells(BBa_K2197502) will have less green fluorescence while comparing it 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 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.


However, due to the problem of missing the operating site in K2197300, which there is no difference of the green fluorescence 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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