Difference between revisions of "Team:Rheda Bielefeld/Interlab"

Latest revision as of 13:46, 16 October 2018

Tables

Fig. 1: The measured Abs600 in relativity to the particle count

Fig. 2: Particle Standard Curve in a logarithm scale

Fig. 3: The measured flourescence per flourescein concentration

Fig. 4: Flourescein Standard Curve in a logarithm scale

InterLab Study

Background

The InterLab Study of this year aims to analyze the connection between the measurement of the Optical Density of E. coli and the GFP in the culture. This proportionality has differed in labs around the world in the past years and we are now trying to approach the results by using the protocol provided by iGEM. This year's major question was: “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-farming units (CFUs) instead of OD?“

Calibrations

At first, we measured the OD600 Reference point according to the LUDOX-Protocol. This included registering the absorbance of LUDOX CL.X and ddH2O at 600 nm.

Partide Standard Curve

For the Partide standard curve, we followed the Microsphere-Protocol, for which we started by mixing 96 µl of microspheres with 904 µl of ddH2O forming the microsphere stock solution. In the next step, we performed a serial dilution of this stock solution with 4 technical replicates through 11 wells each. After this, we measured the Abs600 of all samples, collected in the following table.

Fluorescence Standard Curve

Next, to form the Fluorescence Standard Curve, we executed the Fluorescein-Protocol, according to which we started by resuspending the provided fluorescein in 1 ml of 1xPBS, resulting in a 10xfluorecein stock solution. To lower the concentration of fluorescein we added 100 µl of the 10xfluorescein stock solution afterward to 900 µl of 1xPBS, making it a 1xfluorescein stock solution. Following this, we again performed a serial dilution of 4 technical replicates through 11 wells, after which we measured the fluorescence of all samples.

Cell Measurement Protocol

For completing the Cell Measurement-Protocol we began by transforming 6 different devices, containing plasmids which are all found in pSB1C3, of E. coli DH5 as well as a positive and a negative control on a plate. After letting the plates grow overnight we picked two colonies from each plate which we inoculated in 5-10 ml of LB medium and Cloramphenicol. We inoculated for 16-18 hours at 37 °C and 220 rpm. The next day we added 0.5 ml of each culture to 4.5 ml of LB and Chlor. we diluted to a 1:10 dilution every of which we measured the Abs600 values. We then put 500 µl of every sample at hour 0 on the ice and then inoculated the remainder of the cultures for another 6 hours. At this point, we also put 500µl of each culture in hour 6 on ice. Finally, we measured the fluorescence as well as the Abs600 of both colonies of all 6 devices and the positive and negative control at hour 0 and hour 6.

Colony Forming Units per 0.1 OD600 E.coli cultures

To prepare the Starting Sample we formed overnight cultures of two positive and two negative control cultures diluted 1:8 in LB+Cam. Each culture included 200 µl and was pipetted into a 96-Well plate as well as 2 200µl wells with Blank Media. In the next step, the cultures were diluted to OD600=0.1 in 1 ml of LB+Cam with 3 technical replicates of each well filled with positive and negative control cultures. These formed the Starting Samples.

Next, we performed the dilution series: first we diluted the Starting Samples 1:20 into Dilution 1. We then diluted this dilution 1:20 into Dilution 2. The third dilution was formed by diluting 1xDilution 2 into 20x LB+Cam. This dilution with the final dilution factor 8x10 4 was spread onto an LB+Cam plate. After this, we diluted Dilution 3 1:10 into Dilution 4 which was afterwards also spread on a plate. This culture had the final dilution factor 8x10 5. Lastly, we diluted Dilution 4 once more 1:10. We also spread the hereby formed Dilution 5 (final dilution factor 8x10 6)on a plate. All 36 (12x3) plates were then incubated at 37°C for 20 hours.

After these steps, we counted all colonies on every plate and multiplied these counts by the final dilution factor of each plate.

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-<img src="https://static.igem.org/mediawiki/2018/thumb/a/a3/T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg/320px-T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg">
+<img src="https://static.igem.org/mediawiki/2018/thumb/8/82/T--Rheda_Bielefeld--bacteria%28InterLab%2Cplates%29.jpeg/800px-T--Rheda_Bielefeld--bacteria%28InterLab%2Cplates%29.jpeg" width="45%">
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   <h2> Tables </h2>
-<img src="https://static.igem.org/mediawiki/2018/thumb/a/a3/T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg/320px-T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg" height="200px" width="400px">
+<img src="https://static.igem.org/mediawiki/2018/thumb/a/a3/T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg/320px-T--Rheda_Bielefeld--Particle_Standard_Curve.jpeg" style="height:auto; width:100%">
+<br><br>
+Fig. 1: The measured Abs600 in relativity to the particle count
+<br><br>
+<img src="https://static.igem.org/mediawiki/2018/thumb/9/9b/T--Rheda_Bielefeld--Particle_Standard_Curve%28logscale%29.jpeg/320px-T--Rheda_Bielefeld--Particle_Standard_Curve%28logscale%29.jpeg" style="height:auto; width:100%">
+<br><br>
+Fig. 2: Particle Standard Curve in a logarithm scale
+<br><br>
+<img src="https://static.igem.org/mediawiki/2018/thumb/f/f3/T--Rheda_Bielefeld--Flourescence_Standard_Curve.jpeg/320px-T--Rheda_Bielefeld--Flourescence_Standard_Curve.jpeg" style="height:auto; width:100%">
+<br><br>
+Fig. 3: The measured flourescence per flourescein concentration
+<br><br>
+<img src="https://static.igem.org/mediawiki/2018/thumb/d/d7/T--Rheda_Bielefeld--Flourescence_Standard_Curve%28logscale%29.jpeg/320px-T--Rheda_Bielefeld--Flourescence_Standard_Curve%28logscale%29.jpeg" style="height:auto; width:100%">
+<br><br>
+Fig. 4: Flourescein Standard Curve in a logarithm scale
 </div>
@@ Line 22: / Line 36: @@
 <article>
 <br>
-<h3>Backgrounds</h3>
+<h3>Background</h3>
 <br>
-This years InterLab study aims to analyze the connection between the measurement of the Optical Density of E. coli and the GFP in the culture. This proportionality has differed in labs around the world in the past years and we are now trying to approach the results by using the protocol provided by iGEM. This years big question was: “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-farming units (CFUs) instead of OD?“
+The InterLab Study of this year aims to analyze the connection between the measurement of the Optical Density of E. coli and the GFP in the culture. This proportionality has differed in labs around the world in the past years and we are now trying to approach the results by using the protocol provided by iGEM. This year's major question was: “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-farming units (CFUs) instead of OD?“
 <br><br>
 <h3>Calibrations</h3>
 <br>
-Firstly we measured the OD600 Reference point according to  the LUDOX-Protocol. This included registering the absorbance of LUDOX CL.X and ddH2O at 600 nm
+At first, we measured the OD600 Reference point according to the LUDOX-Protocol. This included registering the absorbance of LUDOX CL.X and ddH2O at 600 nm.
 <br><br>
 <h3>Partide Standard Curve</h3>
 <br>
-For the Partide standard curve we followed the Microsphere-Protocol, for which we started by mixing 96 µl of microspheres with 904 µl of ddH2O forming the microsphere stock solution. In the next step we performed a serial dilution of this stock solution with 4 technical replicates through 11 wells each. After this we measured the Abs600 of all samples, collected in the following table.
+For the Partide standard curve, we followed the Microsphere-Protocol, for which we started by mixing 96 µl of microspheres with 904 µl of ddH2O forming the microsphere stock solution. In the next step, we performed a serial dilution of this stock solution with 4 technical replicates through 11 wells each. After this, we measured the Abs600 of all samples, collected in the following table.
 <br><br>
 <h3>Fluorescence Standard Curve</h3>
 <br>
-Next, to form the Fluorescence Standard Curve, we executed the Fluorescein-Protocol, according to which we started by resuspending the provided fluorescein in 1 ml of 1xPBS, resulting in a 10xfluorecein stock solution. To lower the concentration of fluorescein we then added 100 µl of the 10xfluorescein stock solution to 900 µl of 1xPBS, making it a 1xfluorescein stock solution. Following this we again performed a serial dilution of 4 technical replicates  through 11 wells, after which  we measured the fluorescence of all samples.
+Next, to form the Fluorescence Standard Curve, we executed the Fluorescein-Protocol, according to which we started by resuspending the provided fluorescein in 1 ml of 1xPBS, resulting in a 10xfluorecein stock solution. To lower the concentration of fluorescein we added 100 µl of the 10xfluorescein stock solution afterward to 900 µl of 1xPBS, making it a 1xfluorescein stock solution. Following this, we again performed a serial dilution of 4 technical replicates through 11 wells, after which we measured the fluorescence of all samples.
 <br><br>
 <h3>Cell Measurement Protocol</h3>
 <br>
-For completing the Cell Measurement-Protocol we began by transforming 6 different devices, containing plasmids which are all found in pSB1C3, of E. coli DH5 as well as a positive and a negative control on a plate.
+For completing the Cell Measurement-Protocol we began by transforming 6 different devices, containing plasmids which are all found in pSB1C3, of E. coli DH5 as well as a positive and a negative control on a plate. After letting the plates grow overnight we picked two colonies from each plate which we inoculated in 5-10 ml of LB medium and Cloramphenicol. We inoculated for 16-18 hours at 37 °C and 220 rpm. The next day we added 0.5 ml of each culture to 4.5 ml of LB and Chlor. we diluted to a 1:10 dilution every of which we measured the Abs600 values. We then put 500 µl of every sample at hour 0 on the ice and then inoculated the remainder of the cultures for another 6 hours. At this point, we also put 500µl of each culture in hour 6 on ice. Finally, we measured the fluorescence as well as the Abs600 of both colonies of all 6 devices and the positive and negative control at hour 0 and hour 6.
-After letting the plates grow overnight we picked two colonies from each plate which we inoculated in 5-10 ml of LB medium and Cloramphenicol. We inoculated for 16-18 hours at 37 °C and 220 rpm.
-The next day by adding 0.5 ml of each culture to 4.5 ml of LB and Chlor. we diluted to a 1:10 dilution every of which we measured the Abs600 values. We then put 500 µl of every sample at hour 0 on ice and then inoculated the remainder of the cultures for another 6 hours. A this point we also put 500µl of each culture in hour 6 on ice. Lastly we measured the fluorescence as well as the Abs600 of both colonies of all 6 devices and the positive and negative control at hour 0 and hour 6.
 <br><br>
 <h3>Colony Forming  Units per 0.1 OD600 E.coli cultures</h3>
 <br>
-To prepare the Starting Sample  we formed overnight cultures of two positive and two negative control cultures diluted 1:8 in LB+Cam. Each culture included 200 µl and was pipetted into a 96-Well plate as well as 2 200µl wells with Blank Media. In the next step the cultures were diluted to OD600=0.1 in 1 ml of LB+Cam with 3 technical replicates of each well filled with positive and negative control  cultures. These formed the Starting Samples.
+To prepare the Starting Sample we formed overnight cultures of two positive and two negative control cultures diluted 1:8 in LB+Cam. Each culture included 200 µl and was pipetted into a 96-Well plate as well as 2 200µl wells with Blank Media. In the next step, the cultures were diluted to OD600=0.1 in 1 ml of LB+Cam with 3 technical replicates of each well filled with positive and negative control cultures. These formed the Starting Samples. <br/> <br/>
-<br>
-Next we performed the dilution series: first we diluted the Starting Samples 1:20 into Dilution 1. We then diluted this dilution 1:20 into Dilution 2. The third dilution was formed by diluting 1xDilution 2 into 20x LB+Cam. This dilution with the final dilution factor 8x10  4 was spread onto a LB+Cam plate. After this we diluted Dilution 3 1:10 into Dilution 4 which was afterwards also spread on a plate. This culture had the final dilution factor 8x10 5. Lastly we diluted Dilution 4 once more 1:10. We also spread the hereby formed Dilution 5 (final dilution factor 8x10 6)on an plate. All 36 (12x3) plates were then incubated at 37°C for 20 hours.
+Next, we performed the dilution series: first we diluted the Starting Samples 1:20 into Dilution 1. We then diluted this dilution 1:20 into Dilution 2. The third dilution was formed by diluting 1xDilution 2 into 20x LB+Cam. This dilution with the final dilution factor 8x10 4 was spread onto an LB+Cam plate. After this, we diluted Dilution 3 1:10 into Dilution 4 which was afterwards also spread on a plate. This culture had the final dilution factor 8x10 5. Lastly, we diluted Dilution 4 once more 1:10. We also spread the hereby formed Dilution 5 (final dilution factor 8x10 6)on a plate. All 36 (12x3) plates were then incubated at 37°C for 20 hours. <br/> <br/>
-<br>
-After these steps we counted all colonies on every plate and multiplied these counts by the final dilution factor of each plate.
+After these steps, we counted all colonies on every plate and multiplied these counts by the final dilution factor of each plate.
 </article>
 </div>
- <div class="column right">
+<div class="column right">
+  <img src="https://static.igem.org/mediawiki/2018/thumb/3/38/T--Rheda_Bielefeld--OD600-measurement%28InterLab%2C_e%2Cj%2Cf%2C%29.jpeg/450px-T--Rheda_Bielefeld--OD600-measurement%28InterLab%2C_e%2Cj%2Cf%2C%29.jpeg" style="height:auto; width:100%">
 </div>