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 and BBa_K2197500 are valid. Protocols are included in the protocol page and we will talk about the design and the result 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>
 
  
<p>The aim of doing these assays is to test whether part BBa_K2197300, BBa_K2197400 and BBa_K2197500 are valid. We expect there will be a positively proportional trend when part BBa_K2197300 is put into different concentrations of uric acid. The higher the concentration is, the more GFP will be expressed.   
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<p><font size="4">Design</font></p>
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 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</p>
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<p><font size="4">Steps and Result</font></p>
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<p style="text-align:left !important;">We first incubate the cells in 96 well plate for 1.5 hours and then pipette them into different concentrations of uric acid. After that, 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;">The result is shown as below (Click to see raw data). </p>
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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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<p style="text-align:left !important;">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.
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</p><br>
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<h3 class="sectiontitle">Assay B</h3>
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                        <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 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>
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<p style="text-align:left !important;">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 5 hours and then pipette them into the 96-well plate. After that, 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;">The result is shown as below (Click to see raw data). </p>
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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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<p style="text-align:left !important;">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.
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</p><br>
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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 cells 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 of GFT thus the cell can not fluoresce. 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>
 
  
 
<p style="text-align:left !important;">General Steps:</p>
 
<p style="text-align:left !important;">General Steps:</p>

Revision as of 11:48, 31 October 2017

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 and BBa_K2197500 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 incubate the cells in 96 well plate for 1.5 hours and then pipette them into different concentrations of uric acid. 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).


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 5 hours 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).

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.


General Steps:

  1. Centrifuge 4 ml of cells and discard the LB
  2. Resuspend them with 200ul of PBS
  3. Pipette 50ul of water into A4-A12
  4. Pipette 50 ul of 1x10^-4 mg/dL uric acid into B4-B12
  5. Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:
  6. 123456789101112Concentration of Uric Acid
    A0
    B1x10^-4
    C2x10^-4
    D4x10^-4
    E6x10^-4
    F8x10^-4
    G1x10^-3
    H1x10^-3
  7. 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
  8. Add 50 ul of cultured Cells ( K2197300) into Column 7-12
  9. Let it set for 1-2 hours
  10. Place it in the plate reader to measure the Green Fluorescein

Results

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) 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.

Calibration

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.

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.