Difference between revisions of "Team:Hong Kong-CUHK/InterLab"

 
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<h3>Background</h3>
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<p><h3>Background</h3></p>
−
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.  
+
<p>
 +
<p style="font-family: roboto;font-size:115%;">Reliable and repeatable measurement is the golden rule of engineering, and so does synthetic biology. However, most of the fluorescent measurement data generated nowadays cannot be compared because it is usually reported in relative unit, but not in absolute unit. In addition, different groups may perform measurement with different protocols, which makes it hard to reproduce. Therefore, iGEM developed 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 Study protocol also unifies the measurement procedures and prevents different data processing for the measurement.  
 +
</p>
  
−
<h3>The Fourth InterLab</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.
 
  
−
<h3>Experiment</h3>
+
<p><h3>The Fourth InterLab Study</h3></p>
−
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.
+
 
 +
<p style="font-family: roboto;font-size:115%;">This year, iGEM invited all teams 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 and constructs. We registered and measured all the parts according to the instructions.
 +
</p>
 +
 
 +
<p><h3>Experiments</h3></p>
 +
 
 +
<p style="font-family: roboto;font-size:115%;">iGEM headquarter 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 plasmids. 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.
 +
</p>
 +
 
  
 
<table width="80%">
 
<table width="80%">
 
<thead>
 
<thead>
 
<tr>
 
<tr>
−
<th colspan="6"><u>Summary of parts measured</u></th>
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<th colspan="6"><u>Summary of experimental designs</u></th>
 
</tr>
 
</tr>
 
</thead>
 
</thead>
 +
 
<tr>
 
<tr>
 
<th></th>
 
<th></th>
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<th>Terminator</th>
 
<th>Terminator</th>
 
<th>Backbone</th>
 
<th>Backbone</th>
 +
</tr>
 +
 +
<tr>
 +
<td><b>Negative control (R0040)</b></td>
 +
<td>R0040 (N/A)</td>
 +
<td colspan="3">N/A</td>
 +
<td rowspan="8">pSB1C3</td>
 +
</tr>
 +
 +
<tr>
 +
<td><b>Positive control (I20270)</b></td>
 +
<td>J23151(N/A)</td>
 +
<td>B0032</td>
 +
<td rowspan="7" bgcolor="#40FF00">GFP</td>
 +
<td rowspan="7">B0015</td>
 
</tr>
 
</tr>
  
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<td bgcolor="#FF0000">J23101(1791)</td>
 
<td bgcolor="#FF0000">J23101(1791)</td>
 
<td rowspan="3">B0034</td>
 
<td rowspan="3">B0034</td>
−
<td rowspan="7" bgcolor="#40FF00">GFP</td>
 
−
<td rowspan="7">B0015</td>
 
−
<td rowspan="8">pSB1C3</td>
 
 
</tr>
 
</tr>
  
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</tr>
 
</tr>
  
 +
 +
</table>
 +
 +
<p>
 +
<h3>What is a Bicistronic Device (BCD)?</h3>
 +
</p>
 +
 +
<p><center><img src="https://static.igem.org/mediawiki/2017/0/08/CuhkBCDinstruction.PNG"  style="width:594px;height:216px;"></p></center>
 +
<center><p>Photo adopted from Mutalik, 2013</p></center>
 +
 +
<p>
 +
<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.</p>
 +
 +
<p>
 +
<h3>Method</h3>
 +
</p>
 +
<p>
 +
<p style="font-family: roboto;font-size:115%;">We follow exactly the interlab protocol provided by iGEM(link)
 +
</p>
 +
<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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            <div class="panel panel-default">
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                <div class="panel-heading" role="tab" id="P0">
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                    <h4 class="panel-title">
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                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P0-collapse" aria-expanded="false" aria-controls="P0-collapse">
 +
<div>
 +
                    <div class="col-md-11">Click here to view iGEM Interlab Protocol </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 +
</div>
 +
                    </a>
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                    </h4>
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                </div>
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                <div id="P0-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P0">
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                    <div class="panel-body">               
 +
<ol>
 +
<object type="application/pdf" data="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf" width="100%" height="800"></object >
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</ol>
 +
  </div>
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</div>
 +
</div>
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 +
<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="some-padding"></div>
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<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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            <div class="panel panel-default">
 +
                <div class="panel-heading" role="tab" id="P1">
 +
                    <h4 class="panel-title">
 +
                    <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
 +
<div>
 +
                    <div class="col-md-11">Click here to view our plate reader setting </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
 +
</div>
 +
                    </a>
 +
                    </h4>
 +
 +
                </div>
 +
                <div id="P1-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P1">
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                    <div class="panel-body">               
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<ol>
 +
 +
 +
 +
Microplate used: Corning® clear flat bottom black 96 well plates <br>
 +
Instrument used: BMG LABTECH’s CLARIOstar® <br>
 +
<table width="80%">
 
<tr>
 
<tr>
−
<td><b>Positive control (I20270)</b></td>
+
<th colspan="2">Instrument Settings for measuring OD600 of LUDOX and Cells:</th>
−
<td>J23151(N/A)</td>
+
−
<td>B0032</td>
+
 
</tr>
 
</tr>
  
 
<tr>
 
<tr>
−
<td><b>Negative control (R0040)</b></td>
+
<th colspan="2">Endpoint settings</th>
−
<td>R0040 (N/A)</td>
+
−
<td colspan="3">N/A</td>
+
 
</tr>
 
</tr>
  
−
</table>
+
<tr>
 +
<td>No. of flashes per well</td>
 +
<td>10</td>
 +
</tr>
  
 +
<tr>
 +
<td>Scan mode</td>
 +
<td>orbital averaging</td>
 +
</tr>
  
 +
<tr>
 +
<td>Scan diameter [mm]</td>
 +
<td>3</td>
 +
</tr>
  
−
<h3>What is Bicistronic Device (BCD)?</h3>
+
<tr>
 +
<th colspan="2">Optic settings</th>
 +
<tr>
  
−
<p><img src="https://static.igem.org/mediawiki/2017/0/08/CuhkBCDinstruction.PNG"  style="width:594px;height:216px;"></p>
+
<tr>
 +
<td>Excitation</td>
 +
<td>600</td>
 +
</tr>
  
−
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)
+
<tr>
−
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.
+
<th colspan="2">General settings</th>
 +
</tr>
  
 +
<tr>
 +
<th colspan="2">Top optic used</th>
 +
</tr>
 +
 +
<tr>
 +
<td>Settling time [s]</td>
 +
<td>0.1</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Reading direction</td>
 +
<td>bidirectional, horizontal left to right, top to bottom</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Target temperature [°C]</td>
 +
<td>25</td>
 +
</tr>
 +
</table>
 +
 +
<table width="80%">
 +
<tr>
 +
<th  colspan="2">Instrument Settings for measuring fluorecence of Fluorescein and GFP:</th>
 +
</tr>
 +
 +
<tr>
 +
<th colspan="2">Endpoint settings</th>
 +
</tr>
 +
 +
<tr>
 +
<td>No. of flashes per well</td>
 +
<td>8</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Scan mode</td>
 +
<td>orbital averaging</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Scan diameter [mm]</td>
 +
<td>3</td>
 +
</tr>
 +
 +
<tr>
 +
<th colspan="2">Optic settings</th>
 +
</tr>
 +
 +
<tr>
 +
<td>Excitation:</td>
 +
<td>470-15</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Emission</td>
 +
<td>515-20</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Gain</td>
 +
<td>500</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Focal height [mm]</td>
 +
<td>9</td>
 +
</tr>
 +
 +
<tr>
 +
<th colspan="2">General settings</th>
 +
</tr>
 +
 +
<tr>
 +
<th colspan="2">Top optic used</th>
 +
</tr>
 +
 +
<tr>
 +
<td>Reading direction</td>
 +
<td>bidirectional, horizontal left to right, top to bottom</td>
 +
</tr>
 +
 +
<tr>
 +
<td>Target temperature [°C]</th>
 +
<td>25
 +
</td>
 +
</tr>
 +
 +
</table>
 +
 +
</ol>
 +
 +
</p>
 +
  </div>
 +
</div>
 +
</div>
  
−
<h3>Method</h3>
 
−
We follow exactly the interlab protocol provided by iGEM(link) with the below plate reader setting:
 
 
<br>
 
<br>
−
Microplate used: Corning® clear flat bottom black 96 well plates<br>
 
−
Instrument used: BMG LABTECH’s CLARIOstar®
 
 
<br>
 
<br>
 +
 +
 +
 +
<h3>Results</h3>
 +
 +
<u><p style="font-family: roboto;font-size:115%;">GFP expression in colonies</u> </p>
 +
 +
<p style="font-family: roboto;font-size:115%;">
 +
<img src="https://static.igem.org/mediawiki/2017/0/0a/WhatsApp_Image_2017-10-02_at_9.46.35_PM.jpeg" style="height:400px;" align="left"><br><br>
 +
The colonies were observed from blue light box.<br><br>
 +
The controls show that the plasmid containing GFP gene will give fluorescence.<br><br>
 +
The strength of fluorescence correlates to the strength of constitutive promoters. The higher the strength of promoters, the brighter the colonies. Among the colonies with devices 4-6, the colonies with device 4 showed the highest fluorescence while those with device 6 showed the lowest.
 +
Among the colonies with devices 1-3, the colonies with device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies with devices 1 and 2. </p>
 +
<br><br><br><br><br><br>
 
<br>
 
<br>
−
<b>Instrument Settings for measuring OD600 of LUDOX and Cells:</b>
+
 
−
<br>
+
 
−
<br>
+
<u><p style="font-family: roboto;font-size:115%;">Fluorescence standard curve</u>
−
<b>-   Endpoint settings</b>
+
</p>
−
<br>
+
 
−
No. of flashes per well: 10
+
<p>
−
<br>
+
<center>
−
Scan mode: orbital averaging
+
<img src="https://static.igem.org/mediawiki/2017/5/50/Screen_Shot_2017-10-03_at_5.19.12_PM.png" style="width:600px;height:300px;"></center>
−
<br>
+
<center>
−
Scan diameter [mm]: 3
+
<img src="https://static.igem.org/mediawiki/2017/9/93/Screen_Shot_2017-10-03_at_5.17.03_PM.png" style="width:600px;height:300px;"></center>
−
<br>
+
</p>
−
<br>
+
 
−
<b>-  Optic settings</b>
+
<p>
−
<br>
+
<p style="font-family: roboto;font-size:115%;">
−
Excitation: 600
+
We generated the fluorescence-[fluorescein] standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence from the graphs.
−
<br>
+
−
<br>
+
−
<b>-  General settings</b>
+
−
<br>
+
−
Top optic used<br>
+
−
Settling time [s]: 0.1<br>
+
−
Reading direction: bidirectional, horizontal left to right, top to bottom
+
−
<br>
+
−
Target temperature [°C]: 25
+
−
<br>
+
−
<br>
+
−
<b>Instrument Settings for measuring fluorecence of Fluorescein and GFP:</b>
+
−
<br>
+
−
<br>
+
−
<b>-  Endpoint settings</b>
+
−
<br>
+
−
No. of flashes per well: 8
+
−
<br>
+
−
Scan mode: orbital averaging
+
−
<br>
+
−
Scan diameter [mm]: 3
+
−
<br>
+
−
<br>
+
−
<b>-  Optic settings</b>
+
−
<br>
+
−
Excitation: 470-15
+
−
<br>
+
−
Emission: 515-20
+
−
<br>
+
−
Gain: 500
+
−
<br>
+
−
Focal height [mm]: 9
+
−
<br>
+
−
<br>
+
−
<b>-  General settings
+
−
</b>
+
−
<br>
+
−
Top optic used
+
−
<br>
+
−
Reading direction: bidirectional, horizontal left to right, top to bottom
+
−
<br>
+
−
Target temperature [°C]: 25
+
−
<br>
+
 
</p>
 
</p>
−
<br>
 
−
<br>
 
  
−
<h3>Experiment</h3>
 
 
<br>
 
<br>
−
The colonies was observed from blue light box.
 
−
The controls show that the plasmid containing GFP gene will give fluorescence.
 
 
<br>
 
<br>
−
<br>
 
−
The strength of promoter is correlated to the strength of promoter. The higher the strength, the higher the expression of GFP. 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 <font color="red">no significant difference</font> in fluorescence between the colonies from devices 1 and 2.
 
  
 +
<p style="font-family: roboto;font-size:115%;">
 +
<u>GFP expression in culture</u>
 +
</p>
  
−
<p><img src="https://static.igem.org/mediawiki/2017/1/12/Cuhkigemcolony.PNG"  style="width:400px;height:500px;" ></p>
 
  
−
<p><img src="https://static.igem.org/mediawiki/2017/9/9c/CuhkigemOD600graph.PNG"  style="width:800px;height:400px;" ></p>
+
<p style="font-family: roboto;font-size:115%;">
−
<br><br>
+
We measured the cell density (OD600 values) and fluorescence signal of the cell culture at 2-hour interval. We processed the data by averaging the obtained values from two flasks of culture of different colonies with the same device. The results were represented in the graph below:
 +
</p>
  
 
<p>
 
<p>
−
<b>Fig.1 </b>shows an increasing trend of the cell growth under OD600 absorbance and Fig.2 shows an increasing trend of the fluorescence. <br>
+
<center>
−
<b>Fig.3 </b> shows the trend of fluorescence per cell.<br>
+
<img src="https://static.igem.org/mediawiki/2017/c/c4/Screen_Shot_2017-10-03_at_1.55.24_AM.png"  style="width:600px;height:300px;" >
−
Among devices 4-6, the data match with the fluorescence of their colonies and the strength of their promoters. Device 4 has the highest fluorescence per cell and device 6 has the lowest. Among devices 1-3, the data also match with the fluorescence of their colonies and the strength of their promoters. Device 1 has the highest fluorescence per cell and device 3 has the lowest.  
+
</center>
 +
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2017/8/83/Screen_Shot_2017-10-03_at_1.55.40_AM.png"  style="width:600px;height:300px;" >
 +
</center>
 +
</p>
 +
 
 
<br><br>
 
<br><br>
−
Interestingly, the fluorescence per cell of device 6 and negative control are very close. The fluorescence per cell of device 6 is even lower than the negative control at some time points.
+
 
−
This shows that device 6 produces very little amount of GFP and the result may also be affected by the measurement error in plate reader.
+
<p style="font-family: roboto;font-size:115%;">The increasing trends of OD600 and fluorescence in all devices are shown in the first and the second graph. </p>
 +
 
 
<br><br>
 
<br><br>
−
Another interesting point is the fluorescence per cell of all devices peaked at (t=2hours). One of the possible reasons is the cells spend more energy on cell division but not on producing GFP.
+
 
 +
<p>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2017/e/e5/Screen_Shot_2017-10-03_at_2.05.53_AM.png"  style="width:600px;height:300px;" >
 +
</center>
 
</p>
 
</p>
  
−
<p><img src="https://static.igem.org/mediawiki/2017/e/e9/Cuhkigemfluorescence.PNG"  style="width:500px;height:300px;" ></p>
+
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Among the devices that share the RBS B0034 (devices 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The difference in fluorescence per OD600 is probably due to the promoter strength. Device 1 has the strongest promoter(J23101), thus it gives the highest fluorescence. Device 3 has the weakest promoter(J23117) , thus it gives 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 (devices 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.
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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 has a weaker RBS than B0034.
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Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hour). These may because the increase in cell number is much faster than the overall expression of GFP after t=2 hour.
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1. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60.  
 
1. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60.  
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Latest revision as of 08:49, 31 October 2017





Background

Reliable and repeatable measurement is the golden rule of engineering, and so does synthetic biology. However, most of the fluorescent measurement data generated nowadays cannot be compared because it is usually reported in relative unit, but not in absolute unit. In addition, different groups may perform measurement with different protocols, which makes it hard to reproduce. Therefore, iGEM developed 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 Study protocol also unifies the measurement procedures and prevents different data processing for the measurement.

The Fourth InterLab Study

This year, iGEM invited all teams 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 and constructs. We registered and measured all the parts according to the instructions.

Experiments

iGEM headquarter 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 plasmids. 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.

Summary of experimental designs
Promoter (strength) RBS Coding Sequence Terminator Backbone
Negative control (R0040) R0040 (N/A) N/A pSB1C3
Positive control (I20270) J23151(N/A) B0032 GFP B0015
Device 1 (J364000) J23101(1791) B0034
Device 2 (J364001) J23106(1185)
Device 3 (J364002) J23117(162)
Device 4 (J364003) J23101(1791) BCD2
Device 5 (J364004) J23106(1185)
Device 6 (J364005) J23117(162)

What is a Bicistronic Device (BCD)?

Photo adopted from Mutalik, 2013

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.

Method

We follow exactly the interlab protocol provided by iGEM(link)

    Microplate used: Corning® clear flat bottom black 96 well plates
    Instrument used: BMG LABTECH’s CLARIOstar®
    Instrument Settings for measuring OD600 of LUDOX and Cells:
    Endpoint settings
    No. of flashes per well 10
    Scan mode orbital averaging
    Scan diameter [mm] 3
    Optic settings
    Excitation 600
    General settings
    Top optic used
    Settling time [s] 0.1
    Reading direction bidirectional, horizontal left to right, top to bottom
    Target temperature [°C] 25
    Instrument Settings for measuring fluorecence of Fluorescein and GFP:
    Endpoint settings
    No. of flashes per well 8
    Scan mode orbital averaging
    Scan diameter [mm] 3
    Optic settings
    Excitation: 470-15
    Emission 515-20
    Gain 500
    Focal height [mm] 9
    General settings
    Top optic used
    Reading direction bidirectional, horizontal left to right, top to bottom
    Target temperature [°C] 25



Results

GFP expression in colonies



The colonies were observed from blue light box.

The controls show that the plasmid containing GFP gene will give fluorescence.

The strength of fluorescence correlates to the strength of constitutive promoters. The higher the strength of promoters, the brighter the colonies. Among the colonies with devices 4-6, the colonies with device 4 showed the highest fluorescence while those with device 6 showed the lowest. Among the colonies with devices 1-3, the colonies with device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies with devices 1 and 2.








Fluorescence standard curve

We generated the fluorescence-[fluorescein] standard curves for calibration with the iGEM protocol. The fluorescein concentration can be found with corresponding fluorescence from the graphs.



GFP expression in culture

We measured the cell density (OD600 values) and fluorescence signal of the cell culture at 2-hour interval. We processed the data by averaging the obtained values from two flasks of culture of different colonies with the same device. The results were represented in the graph below:



The increasing trends of OD600 and fluorescence in all devices are shown in the first and the second graph.



Among the devices that share the RBS B0034 (devices 1, 2, 3), device 1 has the highest fluorescence per OD600 and device 3 has the lowest. The difference in fluorescence per OD600 is probably due to the promoter strength. Device 1 has the strongest promoter(J23101), thus it gives the highest fluorescence. Device 3 has the weakest promoter(J23117) , thus it gives 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 (devices 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.


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 has a weaker RBS than B0034.


Another interesting point is the fluorescence per cell of all devices peaked at (t=2 hour). These may because the increase in cell number is much faster than the overall expression of GFP after t=2 hour.




Reference

1. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60.
     

Email: igemcuhk1617@gmail.com