Difference between revisions of "Team:FJNU-China/InterLab"
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<div class="col-xs-12 col-xs-12 col-sm-12" style="width: 95%; margin-left: 40px;"> | <div class="col-xs-12 col-xs-12 col-sm-12" style="width: 95%; margin-left: 40px;"> | ||
| − | <h2 style="color: white;text-shadow: 0 0 20px | + | <h2 style="color: white;text-shadow: 0 0 20px skyblue;">Overview</h2> |
| − | <p> | + | <p> In our project, the InterLab goal is to explore whether we can use standardized absolute cell counts or colony forming units (CFU) instead of OD to reduce lab-to-lab variability in fluorescence measurements.</p> |
<hr> | <hr> | ||
| − | <h2> Materials</h2> | + | <h2 style="color: white;text-shadow: 0 0 20px skyblue;" > Materials</h2> |
<p ><span style="font-size:20px;font-weight:bold;">Strain used</span></br><span style=" font-style:italic;">Escherichia coli</span> DH5α</p> | <p ><span style="font-size:20px;font-weight:bold;">Strain used</span></br><span style=" font-style:italic;">Escherichia coli</span> DH5α</p> | ||
<p ><span style="font-size:20px;font-weight:bold;">Plasmid used</span></br>Negative control: BBa_R0040 Plate 7 Well 2D </br> | <p ><span style="font-size:20px;font-weight:bold;">Plasmid used</span></br>Negative control: BBa_R0040 Plate 7 Well 2D </br> | ||
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| − | <h2 style="color: white;text-shadow: 0 0 20px | + | <h2 style="color: white;text-shadow: 0 0 20px skyblue;">Methods</h2> |
| − | <p> We followed this <a href="https://static.igem.org/mediawiki/2018/3/3e/T--FJNU-China--Interlab-Protocol.pdf">protocol</a> to do the Interlab Study.</br> | + | <p> We followed this <a href="https://static.igem.org/mediawiki/2018/3/3e/T--FJNU-China--Interlab-Protocol.pdf">protocol</a> to do the Interlab Study.</br> All of the calibration measurements have been performed at least three independent experiments. We transformed 8 plasmids into <span style="font-style:italic;">E.coli DH5α</span> competent cells, picked 2 colonies from each plate, and cultured the colonies into 5 mL LB medium supplemented with chloramphenicol when necessary. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0 and 6 hours.</p> |
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<a class="lightbox" href="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png"> | <a class="lightbox" href="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png"> | ||
<img src="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png" alt="figue56"> | <img src="https://static.igem.org/mediawiki/2018/5/56/T--FJNU-China--Interlab-figue56.png" alt="figue56"> | ||
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| + | <a class="lightbox" href="https://static.igem.org/mediawiki/2018/8/8f/T--FJNU-China--interlablast2.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2018/8/8f/T--FJNU-China--interlablast2.png"> | ||
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| − | <h2 style="color: white;text-shadow: 0 0 20px | + | <h2 style="color: white;text-shadow: 0 0 20px skyblue;">Analysis</h2> |
| − | <p style="margin-bottom: 5px;"> A standard curve in a linear relationship can be obtained by calibration experiments.</br> | + | <p style="margin-bottom: 5px;"> A standard curve in a linear relationship can be obtained by calibration experiments. </br> |
| − | By comparing and analyzing the fluorescence values and absorbance | + | By comparing and analyzing the fluorescence values and absorbance values at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences of fluorescence values at 6 h. Among the six different test equipment, the device 4 has the strongest fluorescence, while device 3 has the lowest fluorescence. |
</p> | </p> | ||
<h2 >Discussion</h2> | <h2 >Discussion</h2> | ||
<p style="margin-bottom: 5px;"> During the experiment, we encountered two problems.</br> | <p style="margin-bottom: 5px;"> During the experiment, we encountered two problems.</br> | ||
| − | 1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of 0.02, but there are | + | 1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of OD 0.02, but there are always errors in the actual operation. We cannot accurately dilute the solution to OD 0.02. So we hope that the protocol can provide acceptable error range.</br> |
| − | 2. In the second calibration, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long. | + | 2. In the second calibration experiment, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long. |
| − | + | ||
</p> | </p> | ||
Latest revision as of 03:58, 18 October 2018
Interlab
Overview
In our project, the InterLab goal is to explore whether we can use standardized absolute cell counts or colony forming units (CFU) instead of OD to reduce lab-to-lab variability in fluorescence measurements.
Materials
Strain usedEscherichia coli DH5α
Plasmid usedNegative control: BBa_R0040 Plate 7 Well 2D
Positive control: BBa_I20270 Plate 7 Well 2B
Test Device 1: BBa_J364000 Plate 7 Well 2F
Test Device 2: BBa_J364001 Plate 7 Well 2H
Test Device 3: BBa_J364002 Plate 7 Well 2J
Test Device 4: BBa_J364007 Plate 7 Well 2L
Test Device 5: BBa_J364008 Plate 7 Well 2N
Test Device 6: BBa_J364009 Plate 7 Well 2P
MachineMolecular Devices SpectraMax i3x
Methods
We followed this protocol to do the Interlab Study. All of the calibration measurements have been performed at least three independent experiments. We transformed 8 plasmids into E.coli DH5α competent cells, picked 2 colonies from each plate, and cultured the colonies into 5 mL LB medium supplemented with chloramphenicol when necessary. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0 and 6 hours.
Result
CalibrationCalibration 1: OD600 reference point
Calibration 2: Particle Standard Curve
Cell measurement
Analysis
A standard curve in a linear relationship can be obtained by calibration experiments. By comparing and analyzing the fluorescence values and absorbance values at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences of fluorescence values at 6 h. Among the six different test equipment, the device 4 has the strongest fluorescence, while device 3 has the lowest fluorescence.
Discussion
During the experiment, we encountered two problems. 1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of OD 0.02, but there are always errors in the actual operation. We cannot accurately dilute the solution to OD 0.02. So we hope that the protocol can provide acceptable error range. 2. In the second calibration experiment, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.
Reference
1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182. 2.https://2018.igem.org/Measurement/InterLab 3.http://parts.igem.org/Part:BBa_J61002