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− | <p><h3 | + | <p><h3>Background</h3></p> |
<p> | <p> | ||
− | <font size=" | + | <font size="4">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> |
</p> | </p> | ||
− | <p><h3 | + | <p><h3>The Fourth InterLab</h3></p> |
<p> | <p> | ||
− | <font size=" | + | <font size="4">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> |
</p> | </p> | ||
− | <p><h3 | + | <p><h3>Experiment</h3></p> |
<p> | <p> | ||
− | <font size=" | + | <font size="4">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> |
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− | <h3 | + | <h3>What is Bicistronic Device (BCD)?</h3> |
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− | <h3 | + | <h3>Method</h3> |
</p> | </p> | ||
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− | <h3 | + | <h3>Results</h3> |
</p> | </p> | ||
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− | <u><font size=" | + | <u><font size="5">GFP expression in colonies</font></u> </p> |
<br> | <br> | ||
<p><img src="https://static.igem.org/mediawiki/2017/0/0a/WhatsApp_Image_2017-10-02_at_9.46.35_PM.jpeg" style="width:400px;height:600px;"></p> | <p><img src="https://static.igem.org/mediawiki/2017/0/0a/WhatsApp_Image_2017-10-02_at_9.46.35_PM.jpeg" style="width:400px;height:600px;"></p> | ||
<br> | <br> | ||
<p> | <p> | ||
− | <font size=" | + | <font size="4">The colonies was observed from blue light box. |
The controls show that the plasmid containing GFP gene will give fluorescence.</font> </p> | The controls show that the plasmid containing GFP gene will give fluorescence.</font> </p> | ||
<br> | <br> | ||
<p> | <p> | ||
− | <font size=" | + | <font size="4">The strength of fluorescence is correlated to the strength of promoter. The higher the strength of promoters, the brighter the colonies. Among the colonies from devices 4-6, the colonies from device 4 show the highest fluorescence while those from device 6 show the lowest. |
Among the colonies from devices 1-3, the colonies from device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies from devices 1 and 2.</font> </p> | Among the colonies from devices 1-3, the colonies from device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies from devices 1 and 2.</font> </p> | ||
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− | <u><font size=" | + | <u><font size="5">Fluorescence standard curve</font></u> |
</p> | </p> | ||
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We generated the fluorescein fluorescence standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence using the graph. | We generated the fluorescein fluorescence standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence using the graph. | ||
</font> | </font> | ||
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− | <u><font size=" | + | <u><font size="5">GFP expression in culture</font></u> |
</p> | </p> | ||
<p> | <p> | ||
− | <font size=" | + | <font size="4"> |
We measured the OD600 values and fluorescence signal of the growing culture in fixed time interval. We processed the data by averaging the obtained values of two culture of different colonies with the same device. The results were represented in below graph: | We measured the OD600 values and fluorescence signal of the growing culture in fixed time interval. We processed the data by averaging the obtained values of two culture of different colonies with the same device. The results were represented in below graph: | ||
</font> | </font> | ||
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<br><br> | <br><br> | ||
− | <font size=" | + | <font size="4">The increasing trends of OD600 and fluorescence in all devices are shown in the first and the second graph. </font> |
<br><br> | <br><br> | ||
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− | <font size=" | + | <font size="4"> |
Among the devices that share the RBS B0034 (device 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The different in fluorescence per OD600 is probably due to the strength of promoter. Device 1 has the strongest promoter(J23101), thus it has a highest GFP production rate and give the highest fluorescence. Device 3 has a weakest promoter(J23117) , thus it has a lowest GFP production rate and give the lowest fluorescence. The strength of promoter can also be deduced by comparing the fluorescence per OD600 given by the devices that share the BCD2 (device 4, 5, 6). Device 4 has the strongest fluorescent signal because it uses the strongest promoter (J23101), while device 6 has a weakest fluorescent signal because the weakest promoter(J23117) is used. | Among the devices that share the RBS B0034 (device 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The different in fluorescence per OD600 is probably due to the strength of promoter. Device 1 has the strongest promoter(J23101), thus it has a highest GFP production rate and give the highest fluorescence. Device 3 has a weakest promoter(J23117) , thus it has a lowest GFP production rate and give the lowest fluorescence. The strength of promoter can also be deduced by comparing the fluorescence per OD600 given by the devices that share the BCD2 (device 4, 5, 6). Device 4 has the strongest fluorescent signal because it uses the strongest promoter (J23101), while device 6 has a weakest fluorescent signal because the weakest promoter(J23117) is used. | ||
</font> | </font> | ||
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Comparing the fluorescence signal given by devices with the same promoter but different RBS, we observed that the devices that use BCD2 (devices 4,5 and 6) gave a lower fluorescence comparing with the devices that use B0034 (devices 1, 2 and 3). We may therefore conclude that BCD2 is a weaker RBS than B0034. | Comparing the fluorescence signal given by devices with the same promoter but different RBS, we observed that the devices that use BCD2 (devices 4,5 and 6) gave a lower fluorescence comparing with the devices that use B0034 (devices 1, 2 and 3). We may therefore conclude that BCD2 is a weaker RBS than B0034. | ||
</font> | </font> | ||
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Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hours). These may because the increase in cell number is much faster than the overall expression of GFP after t= 2hrs. | Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hours). These may because the increase in cell number is much faster than the overall expression of GFP after t= 2hrs. | ||
</font> | </font> |
Revision as of 05:25, 17 October 2017