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<h3> <font size="6">Background</font></h3><br> | <h3> <font size="6">Background</font></h3><br> | ||
+ | |||
<font size="3">Reliable and repeatable measurement is the golden rule of engineering, and so do synthetic biology. However, most of the fluorescent measurement data generated nowadays can not be compared, because fluorescence data are usually reported in relative unit, but not in absolute unit. In addition, different groups may perform measurement with different protocol, which makes it hard to reproduce. Therefore, iGEM develop a green fluorescent protein (GFP) measurement protocol in order to produce a more reliable, repeatable measurement of GFP. GFP is one of the most commonly used reporter for measurement and easily to be measured in most of laboratories. In the protocol,the unit for fluorescence data is unified so that the results can be compared. The InterLab protocol also unifies the measurement procedure and prevents different data processing for the measurement. </font> | <font size="3">Reliable and repeatable measurement is the golden rule of engineering, and so do synthetic biology. However, most of the fluorescent measurement data generated nowadays can not be compared, because fluorescence data are usually reported in relative unit, but not in absolute unit. In addition, different groups may perform measurement with different protocol, which makes it hard to reproduce. Therefore, iGEM develop a green fluorescent protein (GFP) measurement protocol in order to produce a more reliable, repeatable measurement of GFP. GFP is one of the most commonly used reporter for measurement and easily to be measured in most of laboratories. In the protocol,the unit for fluorescence data is unified so that the results can be compared. The InterLab protocol also unifies the measurement procedure and prevents different data processing for the measurement. </font> | ||
− | <h3>The Fourth InterLab</h3> | + | <h3><font size="6">The Fourth InterLab</font></h3> |
− | This year, iGEM invited all teams among the world to join the fourth InterLab Study. The aim of the study is to find out how close can the numbers be when fluorescence is measured all around the world using the same InterLab protocol. We registered for the interlab study and measured all the interlab parts using the InterLab plate reader protocol. | + | |
+ | <font size="3>This year, iGEM invited all teams among the world to join the fourth InterLab Study. The aim of the study is to find out how close can the numbers be when fluorescence is measured all around the world using the same InterLab protocol. We registered for the interlab study and measured all the interlab parts using the InterLab plate reader protocol.</font> | ||
+ | |||
+ | <h3><font size="6">Experiment</font></h3> | ||
− | < | + | <font size="3>iGEM provided 8 plasmids for the InterLab Study. Devices 1-6 and positive control have the same reporter gene (GFP), terminator (B0015) and backbone (pSB1C3). However, devices 1-3 share the same RBS (B0034), while devices 4-6 share another modified RBS called bicistronic device (BCD2). Different promoters are also used in different plasmid. According to the strength of promoter described by iGEM2006_Berkeley team, device 1 should have the strongest fluorescence and device 3 should have the weakest among devices 1-3, while Device 4 should have the strongest fluorescence and device 6 should have the weakest among devices 4-6.</font> |
− | iGEM provided 8 plasmids for the InterLab Study. Devices 1-6 and positive control have the same reporter gene (GFP), terminator (B0015) and backbone (pSB1C3). However, devices 1-3 share the same RBS (B0034), while devices 4-6 share another modified RBS called bicistronic device (BCD2). Different promoters are also used in different plasmid. According to the strength of promoter described by iGEM2006_Berkeley team, device 1 should have the strongest fluorescence and device 3 should have the weakest among devices 1-3, while Device 4 should have the strongest fluorescence and device 6 should have the weakest among devices 4-6. | + | |
<table width="80%"> | <table width="80%"> | ||
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− | <h3>What is Bicistronic Device (BCD)?</h3> | + | <h3><font size="6">What is Bicistronic Device (BCD)?</font></h3> |
<p><img src="https://static.igem.org/mediawiki/2017/0/08/CuhkBCDinstruction.PNG" style="width:594px;height:216px;"></p> | <p><img src="https://static.igem.org/mediawiki/2017/0/08/CuhkBCDinstruction.PNG" style="width:594px;height:216px;"></p> | ||
− | Bicistronic device (BCD) is a modified ribosome binding site (RBS) with another cistron. The device consists of another cistron (cistron 1) with another RBS (SD2) | + | <font size="3">Bicistronic device (BCD) is a modified ribosome binding site (RBS) with another cistron. The device consists of another cistron (cistron 1) with another RBS (SD2) |
− | between RBS (SD1) and gene of interest (cistron 2). Also, the stop codon of cistron 1 overlaps the start codon of cistron 2. Ribosome binding efficiency and translation rate will be affected after the secondary structure near the RBS has changed due to the change of gene of interest. This device can maintain the ribosome binding efficiency and translation rate even though the gene of interest has changed. Therefore, it is used to control the amount of fluorescence in this study. BCD is expected to generate a more reliable and precise gene expression. | + | between RBS (SD1) and gene of interest (cistron 2). Also, the stop codon of cistron 1 overlaps the start codon of cistron 2. Ribosome binding efficiency and translation rate will be affected after the secondary structure near the RBS has changed due to the change of gene of interest. This device can maintain the ribosome binding efficiency and translation rate even though the gene of interest has changed. Therefore, it is used to control the amount of fluorescence in this study. BCD is expected to generate a more reliable and precise gene expression.</font> |
− | <h3>Method</h3> | + | <h3><font size="6">Method</font></h3> |
− | We follow exactly the interlab protocol provided by iGEM(link) with the below plate reader setting: | + | <table width="80%"> |
− | < | + | <thead> |
− | Microplate used: Corning® clear flat bottom black 96 well plates< | + | <tr> |
− | Instrument used: BMG LABTECH’s CLARIOstar® | + | <th colspan="2"><u>We follow exactly the interlab protocol provided by iGEM(link) with the below plate reader setting: </u></th> |
− | < | + | </tr> |
− | < | + | </thead> |
− | < | + | |
− | < | + | <tr> |
− | < | + | <th colspan="2">Microplate used: Corning® clear flat bottom black 96 well plates</th> |
− | < | + | </tr> |
− | < | + | |
− | No. of flashes per well | + | <tr> |
− | < | + | <th colspan="2">Instrument used: BMG LABTECH’s CLARIOstar®</th> |
− | Scan mode | + | </tr> |
− | < | + | |
− | Scan diameter [mm] | + | <tr> |
− | < | + | <th colspan="2">Instrument Settings for measuring OD600 of LUDOX and Cells:</th> |
− | < | + | </tr> |
− | < | + | |
− | < | + | <tr> |
− | Excitation | + | <th colspan="2">Endpoint settings</th> |
− | < | + | </tr> |
− | < | + | |
− | < | + | <tr> |
− | < | + | <th>No. of flashes per well</th> |
− | Top optic used< | + | <th>10</th> |
− | Settling time [s] | + | </tr> |
− | Reading direction | + | |
− | < | + | <tr> |
− | Target temperature [°C] | + | <th>Scan mode</th> |
− | < | + | <th>orbital averaging</th> |
− | < | + | </tr> |
− | < | + | |
− | < | + | <tr> |
− | < | + | <th>Scan diameter [mm]</th> |
− | < | + | <th>3</th> |
− | < | + | </tr> |
− | No. of flashes per well | + | |
− | < | + | <tr> |
− | Scan mode | + | <th colspan="2">Optic settings</th> |
− | < | + | <tr> |
− | Scan diameter [mm] | + | |
− | < | + | <tr> |
− | < | + | <th>Excitation</th> |
− | < | + | <th>600</th> |
− | < | + | </tr> |
− | Excitation: 470-15 | + | |
− | < | + | <tr> |
− | Emission | + | <th colspan="2">General settings</th> |
− | < | + | </tr> |
− | Gain | + | |
− | < | + | <tr> |
− | Focal height [mm] | + | <th colspan="2">Top optic used</th> |
− | < | + | </tr> |
− | < | + | |
− | < | + | <tr> |
− | </ | + | <th>Settling time [s]</th> |
− | < | + | <th>0.1</th> |
− | Top optic used | + | </tr> |
− | < | + | |
− | Reading direction | + | <tr> |
− | < | + | <th>Reading direction</th> |
− | Target temperature [°C] | + | <th>bidirectional, horizontal left to right, top to bottom</th> |
− | < | + | </tr> |
− | < | + | |
− | < | + | <tr> |
− | < | + | <th>Target temperature [°C]</th> |
+ | <th>25</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Instrument Settings for measuring fluorecence of Fluorescein and GFP:</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Endpoint settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>No. of flashes per well</th> | ||
+ | <th>8</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan mode</th> | ||
+ | <th>orbital averaging</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Scan diameter [mm]</th> | ||
+ | <th>3</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Optic settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Excitation:</th> | ||
+ | <th>470-15</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Emission</th> | ||
+ | <th>515-20</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Gain</th> | ||
+ | <th>500</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Focal height [mm]</th> | ||
+ | <th>9</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">General settings</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th colspan="2">Top optic used</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Reading direction</th> | ||
+ | <th>bidirectional, horizontal left to right, top to bottom</th> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <th>Target temperature [°C]</th> | ||
+ | <th>25 | ||
+ | </th> | ||
+ | </tr> | ||
<h3>Experiment</h3> | <h3>Experiment</h3> |
Revision as of 11:42, 29 September 2017