Difference between revisions of "Team:Tuebingen/Measurement"

 
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<h1>InterLab Study</h1>
 
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<p>Fluorescence measurement is a method abundant in all major labs.</p>
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            <p> Fluorescence measurement is a method abundant in all major labs. </p>
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<p>Following up on the past years’ success, the iGEM HQ again challenged teams to take part in the InterLab study.  
 
<p>Following up on the past years’ success, the iGEM HQ again challenged teams to take part in the InterLab study.  
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<p> Materials and Methods</p>
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<p>The following five constructs from the InterLab kit were used to transform E. coli DH10beta:
 
<p>The following five constructs from the InterLab kit were used to transform E. coli DH10beta:
 
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<li>Positive Control</li>
 
<li>Positive Control</li>
 
<li>Negative control</li>
 
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<li>Device 2: J23106+I13504</li>
 
<li>Device 2: J23106+I13504</li>
 
<li>Device 3: J23117+I13504</li>
 
<li>Device 3: J23117+I13504</li>
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FITC and LUDOX standards were measured in a TECAN plate reader, using 96-well plates with transparent bottoms. LUDOX was measured at 600nm with a bandwidth of 9 nm and 25 flashes. FITC was excited at 488 nm (bandwidth 9 nm) and emission was recorded at 520 nm (bandwidth 20nm) with a fixed gain of 50 and 25 flashes. Identical settings were used for measurement of the devices.
 
FITC and LUDOX standards were measured in a TECAN plate reader, using 96-well plates with transparent bottoms. LUDOX was measured at 600nm with a bandwidth of 9 nm and 25 flashes. FITC was excited at 488 nm (bandwidth 9 nm) and emission was recorded at 520 nm (bandwidth 20nm) with a fixed gain of 50 and 25 flashes. Identical settings were used for measurement of the devices.
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<img src="https://static.igem.org/mediawiki/2016/1/11/T--Tuebingen--fig1_fitc_standarg_interlab.png" style="cursor: zoom-in;">  
 
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            <p> Fluorescence and OD600 are measured over time. </p>
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<p>After transformation and overnight culture, all cultures were diluted to an OD600 of 0.2. Fluorescence and OD600 were measured over time. All cultures except those transformed with Device 1 show a logarithmic growth (see fig. 2). However, even though the OD600 over time is lower than that for Device 3, the fluorescence over time shows higher values for Device 1. Device 3 shows values at a similar range as the negative control. The highest fluorescence after six hours can be seen for Device 2, which is the only device with higher values that the positive control.
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<figcaption>OD600 (A) and GFP fluorescence emission (B) over the timecourse of six hours.</figcaption>
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            <p> Results </p>
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<p>The normalised fluorescence was calculated using the spreadsheet given out by the iGEM HQ. As figure 3 shows, Device 1 shows the highest normalised fluorescence for all time points. Device 2 shows lower fluorescence, while Device 3 only shows a very basal fluorescence after six hours.  Both Device 1 and 2 show higher fluorescence than the positive control.
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<img src="https://static.igem.org/mediawiki/2016/4/45/T--Tuebingen--fig3_normalised_fluorescence_final.png" style="cursor: zoom-in;">
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<figcaption>Normalised fluorescence emission for all three devices at the given time point.</figcaption>
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            <p> Disease and symptoms </p>
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<p>All three promoters used in this study were characterised by the iGEM-Team Berkeley in 2006 and showed the following relative strengths:
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<li>J23101 (Device 1): 1791</li>
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<li>J23106 (Device 2): 1185</li>
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<li>J23117 (Device 3):  162</li>
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<p>It was therefore expected that Device 1 showed the highest fluorescence, followed by Device 2 and, with a ten-fold decrease compared to Device 1, Device 3.  Our results reflect these values, even though Device 2 showed a lower value than expected.
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Latest revision as of 21:05, 19 October 2016

InterLab Study

Fluorescence measurement is a method abundant in all major labs.

Following up on the past years’ success, the iGEM HQ again challenged teams to take part in the InterLab study.

Measuring the fluorescence of various gene constructs becomes more and more important during everyday lab work. However, the comparability in different labs is still under debate. Using the fact that teams all over the world participate in iGEM, this offers the perfect opportunity to see how results of the same experiment performed all over the world compare. This year’s aim was to compare the fluorescence yield of five different constructs containing GFP under control of different RBS and promoters. By using a FITC standard curve and LUDOX to correct for errors in OD600 measurements, the results of all teams can be easily compared.

Materials and Methods

The following five constructs from the InterLab kit were used to transform E. coli DH10beta:

  • Positive Control
  • Negative control
  • Device 1: J23101+I13504
  • Device 2: J23106+I13504
  • Device 3: J23117+I13504

FITC and LUDOX standards were measured in a TECAN plate reader, using 96-well plates with transparent bottoms. LUDOX was measured at 600nm with a bandwidth of 9 nm and 25 flashes. FITC was excited at 488 nm (bandwidth 9 nm) and emission was recorded at 520 nm (bandwidth 20nm) with a fixed gain of 50 and 25 flashes. Identical settings were used for measurement of the devices.

All measurements were done as described in the Interlab plate reader protocol (see Methods). Transformations were performed as described here Figure 1 shows the FITC standard curve used for the following calculations.

FITC emission standard curve

Fluorescence and OD600 are measured over time.

After transformation and overnight culture, all cultures were diluted to an OD600 of 0.2. Fluorescence and OD600 were measured over time. All cultures except those transformed with Device 1 show a logarithmic growth (see fig. 2). However, even though the OD600 over time is lower than that for Device 3, the fluorescence over time shows higher values for Device 1. Device 3 shows values at a similar range as the negative control. The highest fluorescence after six hours can be seen for Device 2, which is the only device with higher values that the positive control.

OD600 (A) and GFP fluorescence emission (B) over the timecourse of six hours.

Results

The normalised fluorescence was calculated using the spreadsheet given out by the iGEM HQ. As figure 3 shows, Device 1 shows the highest normalised fluorescence for all time points. Device 2 shows lower fluorescence, while Device 3 only shows a very basal fluorescence after six hours. Both Device 1 and 2 show higher fluorescence than the positive control.

Normalised fluorescence emission for all three devices at the given time point.

Disease and symptoms

All three promoters used in this study were characterised by the iGEM-Team Berkeley in 2006 and showed the following relative strengths:

  • J23101 (Device 1): 1791
  • J23106 (Device 2): 1185
  • J23117 (Device 3): 162

It was therefore expected that Device 1 showed the highest fluorescence, followed by Device 2 and, with a ten-fold decrease compared to Device 1, Device 3. Our results reflect these values, even though Device 2 showed a lower value than expected.