Difference between revisions of "Team:Peking/InterLab"
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| − | <h1 style="font-size: xx-large; color: white"><strong>Interlab</strong></h1> | + | <h1 style="font-size: xx-large; color: white;text-shadow:2px 2px 8px #070707;"><strong>Interlab</strong></h1> |
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<ul style="margin-left: 0px;"> | <ul style="margin-left: 0px;"> | ||
| − | <li><a href="# | + | <li><a href="#p1">Introduction</a></li> |
| + | <li><a href="#p2">Materials</a></li> | ||
| + | <li><a href="#p3">Methods</a></li> | ||
| + | <li><a href="#p4">Results</a></li> | ||
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</section> | </section> | ||
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<br> | <br> | ||
| − | + | The Peking iGEM 2017 team is participating in the Fourth InterLaboratory Measurement Study in synthetic | |
| − | + | biology, along with teams from around the world. We introduced eight plasmids into E. coli K-12 DH5-alpha | |
| − | + | and measured the GFP expression levels using a plate reader.<br><br> | |
| − | + | <h2 id = "p1">Introduction</h2> | |
| − | + | Reliable and repeatable measurement is a key component of synthetic biology. However, there have been few | |
| − | + | opportunities to repeat the same measurements in different labs. In order to quantify the degree of | |
| − | + | variability exhibited by engineered genetic constructs across different laboratories, the measurement | |
| − | + | committee invited all iGEM teams to participate in the Interlab study, which provides researchers with a | |
| − | + | detailed protocol and data analysis form, with the aim to produce common, comparable units for measuring GFP | |
| − | + | on different plate readers.<br><br> | |
| − | + | <h2 id = "p2">Materials</h2> | |
| − | + | <br> | |
| − | + | <h3>Plasmids</h3> | |
| − | + | <ol start="1"> | |
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| − | + | <li>Positive Control (BBa_I20270): well 21B</li> | |
| − | + | <li>Negative Control (BBa_R0040): well 21D</li> | |
| − | + | <li>Test Device 1 (BBa_J364000): well 21F</li> | |
| − | + | <li>Test Device 2 (BBa_J364001): well 21H</li> | |
| − | + | <li>Test Device 3 (BBa_J364002): well 21J</li> | |
| − | + | <li>Test Device 4 (BBa_J364003): well 21L</li> | |
| − | + | <li>Test Device 5 (BBa_J364004): well 21N</li> | |
| − | + | <li>Test Device 6 (BBa_J364005): well 21P</li> | |
| − | + | </ol> | |
| − | + | <br> | |
| − | + | <h3>Strains</h3> | |
| − | + | Escherichia coli strain DH5α<br> | |
| − | + | <br> | |
| − | + | <h3>Reagent</h3> | |
| − | + | <div class="nonumberitem"> | |
| − | + | <ul> | |
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| − | <a class="mdl-button mdl-js-button mdl-button--raised mdl-js-ripple-effect" href = "https://2017.igem.org/Team:Peking/Lab" style="background-color: #CB2C32; color: white;"> | + | |
| + | <li class="nonumberitem">1ml LUDOX</li> | ||
| + | <li class="nonumberitem">ddH<SUB>2</SUB>O</li> | ||
| + | <li class="nonumberitem">Fluorescein<br></li> | ||
| + | <li class="nonumberitem">10ml 1xPBS<br></li> | ||
| + | </ul> | ||
| + | </div> | ||
| + | |||
| + | <br> | ||
| + | <h3>Instruments</h3> | ||
| + | <div class="nonumberitem"> | ||
| + | <ul> | ||
| + | |||
| + | |||
| + | <li>Pipettes</li> | ||
| + | <li>96-well plate</li> | ||
| + | <li>50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)</li> | ||
| + | <li>1.5-ml Eppendorf tubes for sample storage, ice bucket with ice</li> | ||
| + | </ul> | ||
| + | </div> | ||
| + | <br> | ||
| + | <h2 id = "p3">Methods</h2> | ||
| + | At first, we used LUDOX-S40 as a single-point reference to obtain a ratiometric conversion factor to | ||
| + | transform our absorbance readings into standard OD600 data. Simultaneously, the ratiometric conversion factor accounts for instrument differences.<br><br> | ||
| + | Secondly, we generated a standard curve of fluorescence as a function of fluorescein concentration. We can thus use the standard curve to convert the fluorescence of GFP into a concentration of GFP.<br><br> | ||
| + | |||
| + | Last and most importantly, we measured the A600 and fluorescence of green fluorescent protein. The GFP is used as a measurement marker of promoter activity, and the fluorescence/OD600 ratio is used to give an adjustment of the relative expression per cell. Furthermore, we tested some RBS devices that are intended to make gene expression more precise and reliable. | ||
| + | <br><br> | ||
| + | |||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/31/Peking_interlab_protocol1.jpeg" | ||
| + | height="400px"/><br> | ||
| + | <br> | ||
| + | <strong>Figure1. The 96 well plate of the a dilution series of fluorescein in 4 replicates.</strong><br> | ||
| + | <br><br> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/3/3d/Peking_interlab_protocol2.jpeg" | ||
| + | height="400px"/><br> | ||
| + | <br> | ||
| + | <strong>Figure2. The cultures at 6 hours of incubation.</strong><br> | ||
| + | <br><br> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c3/Peking_interlab_protocol3.jpeg" | ||
| + | height="400px"/><br> | ||
| + | <br> | ||
| + | <strong>Figure3. The machine and materials of the interlab study.</strong><br> | ||
| + | <br><br> | ||
| + | |||
| + | <br><br> | ||
| + | |||
| + | <h2 id = "p4">Results</h2> | ||
| + | Samples were prepared according to the standard protocol.The results for E. coli DH5α are shown in | ||
| + | tables 1-10.<br> | ||
| + | <br> | ||
| + | |||
| + | <h3>OD600 reference point</h3><br> | ||
| + | <strong>Table1. OD600 reference point.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f5/Peking_interlab_data1.png" | ||
| + | height="400px"/><br> | ||
| + | <br><br> | ||
| + | |||
| + | <h3>Fluorescence standard curve</h3><br> | ||
| + | <strong>Table2. Fluorescein standard curve.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/46/Peking_interlab_data2.png" | ||
| + | height="400px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Figure4. Fluorescein standard curve.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/af/Peking_interlab_curve1.png" | ||
| + | height="255px"/><br> | ||
| + | <br><br> | ||
| + | |||
| + | <strong>Figure5. Fluorescein standard curve (log scale).</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/09/Peking_interlab_curve2.png" | ||
| + | height="250px"/><br> | ||
| + | <br><br> | ||
| + | |||
| + | <h3>Raw Plate Reader Measurements</h3><br> | ||
| + | <strong>Table3. Raw Plate Reader Measurements of Fluorescence Raw at 0 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/0c/Peking_interlab_data3.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table4. Raw Plate Reader Measurements of Fluorescence Raw at 2 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/ea/Peking_interlab_data4.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table5. Raw Plate Reader Measurements of Fluorescence Raw at 4 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/8/8c/Peking_interlab_data5.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table6. Raw Plate Reader Measurements of Fluorescence Raw at 6 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/e5/Peking_interlab_data6.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <h3>Cell Measurement</h3> | ||
| + | |||
| + | <strong>Table7. Raw data of Abs600 measurement at 0 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/0a/Peking_interlab_data7.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table8. Raw data of Abs600 measurement at 2 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/8/8b/Peking_interlab_data8.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table9. Raw data of Abs600 measurement at 4 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/6e/Peking_interlab_data9.png" | ||
| + | height="300px"/><br> | ||
| + | <br> | ||
| + | |||
| + | <strong>Table10. Raw data of Abs600 measurement at 6 Hour.</strong><br> | ||
| + | <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/5/59/Peking_interlab_data10.png" | ||
| + | height="300px"/><br> | ||
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| − | <h2 style="color: white; "> <strong>Peking iGEM</strong> 2017</h2> | + | <h2 style="color: white; "><strong>Peking iGEM</strong> 2017</h2> |
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| − | <li style="color: white; line-height: 1.88em;">Peking University,<br> No.5 Yiheyuan Road Haidian District, Beijing, P.R.China<br>100871</li> | + | <li style="color: white; line-height: 1.88em;">Peking University,<br> No.5 Yiheyuan Road Haidian |
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Latest revision as of 13:40, 1 November 2017
Interlab
The Peking iGEM 2017 team is participating in the Fourth InterLaboratory Measurement Study in synthetic biology, along with teams from around the world. We introduced eight plasmids into E. coli K-12 DH5-alpha and measured the GFP expression levels using a plate reader.
Introduction
Reliable and repeatable measurement is a key component of synthetic biology. However, there have been few opportunities to repeat the same measurements in different labs. In order to quantify the degree of variability exhibited by engineered genetic constructs across different laboratories, the measurement committee invited all iGEM teams to participate in the Interlab study, which provides researchers with a detailed protocol and data analysis form, with the aim to produce common, comparable units for measuring GFP on different plate readers.Materials
Plasmids
- Positive Control (BBa_I20270): well 21B
- Negative Control (BBa_R0040): well 21D
- Test Device 1 (BBa_J364000): well 21F
- Test Device 2 (BBa_J364001): well 21H
- Test Device 3 (BBa_J364002): well 21J
- Test Device 4 (BBa_J364003): well 21L
- Test Device 5 (BBa_J364004): well 21N
- Test Device 6 (BBa_J364005): well 21P
Strains
Escherichia coli strain DH5αReagent
- 1ml LUDOX
- ddH2O
- Fluorescein
- 10ml 1xPBS
Instruments
- Pipettes
- 96-well plate
- 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
- 1.5-ml Eppendorf tubes for sample storage, ice bucket with ice
Methods
At first, we used LUDOX-S40 as a single-point reference to obtain a ratiometric conversion factor to transform our absorbance readings into standard OD600 data. Simultaneously, the ratiometric conversion factor accounts for instrument differences.Secondly, we generated a standard curve of fluorescence as a function of fluorescein concentration. We can thus use the standard curve to convert the fluorescence of GFP into a concentration of GFP.
Last and most importantly, we measured the A600 and fluorescence of green fluorescent protein. The GFP is used as a measurement marker of promoter activity, and the fluorescence/OD600 ratio is used to give an adjustment of the relative expression per cell. Furthermore, we tested some RBS devices that are intended to make gene expression more precise and reliable.
Figure1. The 96 well plate of the a dilution series of fluorescein in 4 replicates.
Figure2. The cultures at 6 hours of incubation.
Figure3. The machine and materials of the interlab study.
Results
Samples were prepared according to the standard protocol.The results for E. coli DH5α are shown in tables 1-10.OD600 reference point
Table1. OD600 reference point.
Fluorescence standard curve
Table2. Fluorescein standard curve.
Figure4. Fluorescein standard curve.
Figure5. Fluorescein standard curve (log scale).
Raw Plate Reader Measurements
Table3. Raw Plate Reader Measurements of Fluorescence Raw at 0 Hour.
Table4. Raw Plate Reader Measurements of Fluorescence Raw at 2 Hour.
Table5. Raw Plate Reader Measurements of Fluorescence Raw at 4 Hour.
Table6. Raw Plate Reader Measurements of Fluorescence Raw at 6 Hour.
Cell Measurement
Table7. Raw data of Abs600 measurement at 0 Hour.
Table8. Raw data of Abs600 measurement at 2 Hour.
Table9. Raw data of Abs600 measurement at 4 Hour.
Table10. Raw data of Abs600 measurement at 6 Hour.
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