Difference between revisions of "Team:Aalto-Helsinki/InterLab"

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<br><br>
 
<br><br>
  
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Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 10<sup>8</sup> to 6 ⋅ 10<sup>8</sup> cfu/µg. The transformation efficiency was 6.6 ⋅ 10<sup>5</sup> cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
+
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using <i>Competent Cell Test Kit</i> provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 10<sup>8</sup> to 6 ⋅ 10<sup>8</sup> cfu/µg. The transformation efficiency was 6.6 ⋅ 10<sup>5</sup> cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
 
<br><br>
 
<br><br>
  
 
<font size="1" face="Calibri" >
 
<font size="1" face="Calibri" >
 
  <table style="width:50%">
 
  <table style="width:50%">
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   <caption>Colonies</caption>
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   <caption>Table 1. Colonies</caption>
 
   <tr>
 
   <tr>
 
     <th>DNA concentration [pg/µl]</th>
 
     <th>DNA concentration [pg/µl]</th>
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   </tr>
 
   </tr>
  
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<br>
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<br><br>
  
 
</table>  
 
</table>  
 
  <table style="width:50%">
 
  <table style="width:50%">
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   <caption>Transformation efficiency</caption>
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   <caption>Table 2. Transformation efficiency</caption>
 
   <tr>
 
   <tr>
 
     <th>DNA concentration [pg/µl]</th>
 
     <th>DNA concentration [pg/µl]</th>
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The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.
 
The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.
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Device Part Number
+
 
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Negative control BBa_R0040
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<font size="1" face="Calibri" >
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Positive control BBa_I20270
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<table style="width:50%">
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Test Device 1 BBa_J364000
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  <tr>
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Test Device 2 BBa_J364001
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    <th>Device</th>
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Test Device 3 BBa_J364002
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    <th>Part Number</th>
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Test Device 4 BBa_J364007
+
  </tr>
−
Test Device 5 BBa_J364008
+
  <tr>
−
Test Device 6 BBa_J364009
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    <td>Negative control</td>
 +
    <td>BBa_R0040</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Positive control</td>
 +
    <td>BBa_I20270</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Test Device 1</td>
 +
    <td>BBa_J364000</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 2</td>
 +
    <td>BBa_J364001</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 3</td>
 +
    <td>BBa_J364002</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 4</td>
 +
    <td>BBa_J364007</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 5</td>
 +
    <td>BBa_J364008</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Test Device 6</td>
 +
    <td>BBa_J364009</td>
 +
  </tr>
 +
 
 +
 
 +
 
 +
</font>
 
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Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results.
+
Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD<sub>600</sub> to Abs<sub>600</sub>. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. <i>Table 3</i> shows the results.
 
<br><br>
 
<br><br>
 +
 +
<font size="1" face="Calibri" >
 +
<table style="width:50%">
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  <caption>Table 3. Calibration 1</caption>
 +
  <tr>
 +
    <th></th>
 +
    <th>LUDOX CL-X</th>
 +
    <th>ddH<sub>2</sub>O</th>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 1</td>
 +
    <td>0.051</td>
 +
    <td>0.041</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 2</td>
 +
    <td>0.051</td>
 +
    <td>0.036</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 3</td>
 +
    <td>0.048</td>
 +
    <td>0.036</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Replicate 4</td>
 +
    <td>0.060</td>
 +
    <td>0.038</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Arith. Mean</td>
 +
    <td>0.053</td>
 +
    <td>0.038</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Corrected Abs<sub>600</sub></td>
 +
    <td>0.015</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Reference OD<sub>600</sub></td>
 +
    <td>0.063</td>
 +
  </tr>
 +
  <tr>
 +
    <td>OD<sub>600</sub>/Abs<sub>600</sub></td>
 +
    <td>4.271</td>
 +
  </tr>
 +
</font>
  
 
<font face="calibri" size="4">Particle Standard Curve</font>
 
<font face="calibri" size="4">Particle Standard Curve</font>
 
<br><br>
 
<br><br>
  
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Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results.
+
Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures below show the results.
 
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The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results.
+
The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures below show the results.
 
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Revision as of 09:54, 20 July 2018

Introduction

Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.

Results

Competency

Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.



Table 1. Colonies
DNA concentration [pg/µl] 10 100
Plate 1 10 51
Plate 2 5 89
Plate 3 3 76
Average 6 72
Table 2. Transformation efficiency
DNA concentration [pg/µl] 10 100
Plate 1 1 ⋅ 106 cfu/µg 5.1 ⋅ 105 cfu/µg
Plate 2 5 ⋅ 105 cfu/µg 8.9 ⋅ 105 cfu/µg
Plate 3 3 ⋅ 105 cfu/µg 7.6 ⋅ 105 cfu/µg
Average 6 ⋅ 105 cfu/µg 7.2 ⋅ 105 cfu/µg


Parts

The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.

OD600 Reference Point

Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results.

Device Part Number
Negative control BBa_R0040
Positive control BBa_I20270
Test Device 1 BBa_J364000
Test Device 2 BBa_J364001
Test Device 3 BBa_J364002
Test Device 4 BBa_J364007
Test Device 5 BBa_J364008
Test Device 6 BBa_J364009
Particle Standard Curve

Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures below show the results.




Fluorescence Standard Curve

The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures below show the results.



Cell Measurements

Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process.

Absorbance

Fluorescence

[1] http://parts.igem.org/Help:Protocols/Competent_Cells
[2] http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit
[3] http://parts.igem.org/Help:Protocols/Transformation
[4] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

Table 3. Calibration 1
LUDOX CL-X ddH2O
Replicate 1 0.051 0.041
Replicate 2 0.051 0.036
Replicate 3 0.048 0.036
Replicate 4 0.060 0.038
Arith. Mean 0.053 0.038
Corrected Abs600 0.015
Reference OD600 0.063
OD600/Abs600 4.271