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<p><h3>Background</h3></p> | <p><h3>Background</h3></p> | ||
<p> | <p> | ||
− | < | + | <p style="font-family: roboto;font-size:115%;">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>The Fourth InterLab</h3></p> | <p><h3>The Fourth InterLab</h3></p> | ||
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− | < | + | <p style="font-family: roboto;font-size:115%;">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>Experiment</h3></p> | <p><h3>Experiment</h3></p> | ||
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− | < | + | <p style="font-family: roboto;font-size:115%;">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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− | < | + | <p style="font-family: roboto;font-size:115%;">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.</font> | 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> | ||
</p> | </p> | ||
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− | < | + | <p style="font-family: roboto;font-size:115%;">We follow exactly the interlab protocol provided by iGEM(link)</font> |
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
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− | <u>< | + | <u><p style="font-family: roboto;font-size:115%;">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> | ||
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− | < | + | <p style="font-family: roboto;font-size:115%;">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> | ||
− | < | + | <p style="font-family: roboto;font-size:115%;">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>< | + | <u><p style="font-family: roboto;font-size:115%;">Fluorescence standard curve</font></u> |
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− | < | + | <p style="font-family: roboto;font-size:115%;"> |
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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− | < | + | <p style="font-family: roboto;font-size:115%;"> |
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 17:25, 24 October 2017