Difference between revisions of "Team:Vilnius-Lithuania/InterLab"

 
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         At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the <strong>LUDOX Protocol</strong> in order to obtain a conversion factor to transform absorbance (Abs<sub>600</sub>) from the plate reader into a comparable OD<sub>600</sub> measurement as would be obtained with a spectrophotometer. Next, we completed the <strong>Microsphere Protocol</strong> as it allows a standard curve of particle concentration which is used to convert Abs<sub>600</sub> measurements to an estimated number of cells. Finally, by completing the <strong>Fluorescein Protocol</strong> we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).
 
         At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the <strong>LUDOX Protocol</strong> in order to obtain a conversion factor to transform absorbance (Abs<sub>600</sub>) from the plate reader into a comparable OD<sub>600</sub> measurement as would be obtained with a spectrophotometer. Next, we completed the <strong>Microsphere Protocol</strong> as it allows a standard curve of particle concentration which is used to convert Abs<sub>600</sub> measurements to an estimated number of cells. Finally, by completing the <strong>Fluorescein Protocol</strong> we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).
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        <p></p><strong>Tab. 1</strong> Parts received and tested during iGEM’s fifth InterLab Study
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        <p><strong>Tab. 1</strong> Parts received and tested during iGEM’s fifth InterLab Study</p>
 
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  <p>Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD<sub>600</sub> measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.</p>
 
  <p>Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD<sub>600</sub> measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.</p>
  
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<strong>Tab. 2</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419
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<p><strong>Tab. 2</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419</p>
 
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  <p>Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs<sub>600</sub> measurements to an estimated number of cells.
 
  <p>Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs<sub>600</sub> measurements to an estimated number of cells.
 
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<<img src="https://static.igem.org/mediawiki/2018/3/31/T--Vilnius-Lithuania--1_InterLab.png"
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<img src="https://static.igem.org/mediawiki/2018/3/31/T--Vilnius-Lithuania--1_InterLab.png"/>
 
       <p><strong>Fig. 1</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.</p>
 
       <p><strong>Fig. 1</strong> LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.</p>
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<img src="https://static.igem.org/mediawiki/2018/b/bc/T--Vilnius-Lithuania--2_InterLab.png"/>
 
       <p><strong>Fig. 2</strong> Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.</p>
 
       <p><strong>Fig. 2</strong> Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.</p>
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<div class="image-container">
 
          
 
          
 
<p>During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig.  1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.
 
<p>During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig.  1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.
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<p>In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.</p>
 
<p>In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.</p>
 
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<img src="https://static.igem.org/mediawiki/2018/b/b0/T--Vilnius-Lithuania--3_InterLab.png">
 
<img src="https://static.igem.org/mediawiki/2018/b/b0/T--Vilnius-Lithuania--3_InterLab.png">
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       <strong>Fig. 3</strong> Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.</p>
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       <p><strong>Fig. 3</strong> Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.</p>
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--Vilnius-Lithuania--4_InterLab.png">
 
       <p><strong>Fig. 4</strong> A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.
 
       <p><strong>Fig. 4</strong> A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.
 
</p>
 
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       <p>During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.</p>
 
       <p>During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.</p>
  
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       <p><strong>Fig. 5</strong> Graph comparing the raw Abs<sub>600</sub> prior incubation and at hour 6 for each colony using each control/device</p>
 
       <p><strong>Fig. 5</strong> Graph comparing the raw Abs<sub>600</sub> prior incubation and at hour 6 for each colony using each control/device</p>
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<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Vilnius-Lithuania--6_InterLab.png"
 
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      <p><strong>Fig. 6 </strong>Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device</p>
 
  
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<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Vilnius-Lithuania--6_InterLab.png">
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      <p><strong>Fig. 6 </strong>Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device</p>
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  <p>Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected.  
 
  <p>Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected.  
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<strong>Tab. 3</strong> Colony forming units (CFU) per 1 mL of an OD<sub>600</sub> = 0.1culture
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<p> <strong>Tab. 3</strong> Colony forming units (CFU) per 1 mL of an OD<sub>600</sub> = 0.1culture</p>
  
 
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     <tr>
 
     <tr>
 
         <td>1.1 Positive Control</td>
 
         <td>1.1 Positive Control</td>
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         <td>0.132667</td>
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         <td>0.132667 * 10^8</td>
 
     </tr> <tr>
 
     </tr> <tr>
 
             <td>1.2 Positive Control</td>
 
             <td>1.2 Positive Control</td>
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         <td>0.086667</td>
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         <td>0.086667 * 10^8</td>
 
   </tr> <tr>
 
   </tr> <tr>
 
             <td>1.3 Positive Control</td>
 
             <td>1.3 Positive Control</td>
−
         <td>0.271333</td>
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         <td>0.271333 * 10^8</td>
 
     </tr> <tr>
 
     </tr> <tr>
 
             <td>2.1 Positive Control</td>
 
             <td>2.1 Positive Control</td>
−
         <td>0.448667</td>
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         <td>0.448667 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>2.2 Positive Control</td>
 
             <td>2.2 Positive Control</td>
−
         <td>0.394667</td>
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         <td>0.394667 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>2.3 Positive Control</td>
 
             <td>2.3 Positive Control</td>
−
         <td>0.659667</td>
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         <td>0.659667 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>3.1 Negative Control</td>
 
             <td>3.1 Negative Control</td>
−
         <td>0.236</td>
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         <td>0.236 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>3.2 Negative Control</td>
 
             <td>3.2 Negative Control</td>
−
         <td>0.722</td>
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         <td>0.722 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>3.3 Negative Contro</td>
 
             <td>3.3 Negative Contro</td>
−
         <td>0.346667</td>
+
         <td>0.346667 * 10^8</td>
 
     </tr>  <tr>
 
     </tr>  <tr>
 
             <td>4.1 Negative control</td>
 
             <td>4.1 Negative control</td>
−
         <td>0.494</td>
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         <td>0.494 * 10^8</td>
 
     </tr>  <tr>  
 
     </tr>  <tr>  
 
             <td>4.2 Negative control</td>
 
             <td>4.2 Negative control</td>
−
         <td>0.279</td>
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         <td>0.279 * 10^8</td>
 
     </tr>  <tr>  
 
     </tr>  <tr>  
 
             <td>4.3 Negative control</td>
 
             <td>4.3 Negative control</td>
−
         <td>0.395</td>  
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         <td>0.395 * 10^8</td>  
 
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Latest revision as of 21:42, 4 November 2018

InterLab

Studying Fluorescence

The goal of this year’s InterLab Study was to identify and minimize the sources of systematic variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).

Participating in the fifth iGEM InterLab Study was a great opportunity to start this year’s competition as well as acquire some valuable knowledge which we implemented into practice during the project.

Description

At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the LUDOX Protocol in order to obtain a conversion factor to transform absorbance (Abs600) from the plate reader into a comparable OD600 measurement as would be obtained with a spectrophotometer. Next, we completed the Microsphere Protocol as it allows a standard curve of particle concentration which is used to convert Abs600 measurements to an estimated number of cells. Finally, by completing the Fluorescein Protocol we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Tab 1).

Tab. 1 Parts received and tested during iGEM’s fifth InterLab Study

Device Part Number Features
Negative control BBa_R0040 Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control BBa_I20270 J23151 inserted in the Promoter MeasKit
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter from the Anderson collection

Results and Discussion

1. MEASUREMENT OF LUDOX CL-X OD600 REFERENCE POINT

Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Tab. 2) which will be used in further Interlab study measurements.

Tab. 2 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419

LUDOX CL-X H2O
Negative control BBa_R0040 Medium strength promoter, promoter is constitutive and repressed by TetR
Positive Control BBa_I20270 J23151 inserted in the Promoter MeasKit
Test Device 1 BBa_J364000 GFP expressing constitutive device
Test Device 2 BBa_J364001 GFP expressing constitutive device
Test Device 3 BBa_J364002 GFP expressing constitutive device
Test Device 4 BBa_J364007 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 5 BBa_J364008 Expresses GFP under the control of a constitutive promoter from the Anderson collection
Test Device 6 BBa_J364009 Expresses GFP under the control of a constitutive promoter from the Anderson collection

2. GRAPHING A SILICA MICROSPHERE ABSORBANCE (Abs600) STANDARD CURVE

Monodisperse silica microspheres exhibit size and optical characteristics similar to cells, with the additional benefit that the number of particles in a solution is known. Therefore, this measurement allowed us to construct a standard curve which can be used to convert Abs600 measurements to an estimated number of cells.

Fig. 1 LUDOX CL-X measurement. Obtained ratiometric conversion factor is 3,419.

Fig. 2 Particle standard curve generated by measuring the absorbance of serial dilutions of silica microspheres (known amount of particles per volume) displayed in a log scale to demonstrate a linear relationship between particle count per volume and absorbance.

During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig. 1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.

3. GRAPHING A FLUORESCEIN FLUORESCENCE STANDARD CURVE

In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.

Fig. 3 Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.

Fig. 4 A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.

During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.

Cell Measurements

For cell measurements we used the same settings that we used in our calibration measurements. At first, according to the standard protocol we transformed cells with 8 different plasmids (Tab. 1). We picked 2 colonies from each transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. We grew the cells overnight (16-18 hours) at 37 °C and 220 rpm. After that we diluted the cultures to a target Abs600 of 0.02. We took samples from these diluted cultures prior to incubation and after 6 hours of incubation measured Abs600 (Fig. 5) and fluorescence (Fig. 6).

Fig. 5 Graph comparing the raw Abs600 prior incubation and at hour 6 for each colony using each control/device

Fig. 6 Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device

Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected. Based on the assumption that one bacterial cell gives rise to one colony, colony forming units per 1 mL of an OD600 = 0.1 culture was calculated by counting the colonies on each plate with fewer than 300 colonies and multiplying the colony count by the Final Dilution Factor on each plate The results are shown in Tab. 3.

Tab. 3 Colony forming units (CFU) per 1 mL of an OD600 = 0.1culture

Samples CFU/ml in Starting Sample
1.1 Positive Control 0.132667 * 10^8
1.2 Positive Control 0.086667 * 10^8
1.3 Positive Control 0.271333 * 10^8
2.1 Positive Control 0.448667 * 10^8
2.2 Positive Control 0.394667 * 10^8
2.3 Positive Control 0.659667 * 10^8
3.1 Negative Control 0.236 * 10^8
3.2 Negative Control 0.722 * 10^8
3.3 Negative Contro 0.346667 * 10^8
4.1 Negative control 0.494 * 10^8
4.2 Negative control 0.279 * 10^8
4.3 Negative control 0.395 * 10^8

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