Team:NCTU Formosa/RNA data

From 2011.igem.org

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<div id="blueBox"><p>RNA Thermometer</p></div>
<div id="blueBox"><p>RNA Thermometer</p></div>
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<div id="Box"><h2>Data</h2>
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<div id="Box"><h2> Design </h2>
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<p>We constructed three circuits to make sure the function of RNA thermometer. At the end we will combine those parts in one complete circuit with Low-temperature release system.</p>
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<p>RNA Thermometer system is about an RNA-based system that is able to have differential gene expression when temperature changes. The expression of heat-shock, cold-shock and some virulence genes are coordinated in response to temperature changes. Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is a widely used regulatory strategy. RNA thermometers are thermo-sensors that regulate gene expression by temperature-induced changes in RNA conformation.</p>
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<div><img src = "http://partsregistry.org/wiki/images/b/bb/RNA_data_1.jpg" width="450"></div>
 
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<br><b>Circuit A: </b>
 
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<a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> is a constitutive promoter, and the 37℃induced RBS we use is <a href="http://partsregistry.org/Part:BBa_K115002">BBa_K115002</a>. The mGFP is the gene coding for “Green Fluorescence Protein”.<br><br>
 
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<p>First, we combined a common promoter with 37°C induced RBS (RNA thermometer) and mGFP gene. It is for testing the function of RNA thermometer. Therefore, we can get the RNA thermometer’s regulation on green fluorescent protein expression. The flow cytometer’s data shows the highest fluorescent protein expression at the 37°C and 42°C.</p>
 
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<p>That proves the 37°C induced RBS (RNA thermometer) has an appropriate function.(figure 3.)</p>
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<center><div><img src = "http://partsregistry.org/wiki/images/0/04/RNA_Design_1.jpg"></div></center>
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<br><b>Figure 1:</b> RNA thermometer<br>At the left, the RNA forms stable base-pairs on the Shine-Dalgarno sequence (SD sequence), disabling the ribosome to bind. (SD sequence is the polypurine sequence AGGAGG centered about 10 bp before the AUG initiation codon on bacterial mRNA.) It is now in the on-state. The base-pairing of this RNA region will block the expression of the protein encoded behind it. In the other hand, at the right, certain external factor is added and causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state.<br><br>
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<div><img src = "http://partsregistry.org/wiki/images/b/bf/RNA_data_1-2.jpg" width="700"></div>
 
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<br><b>Figure 3: Testing RNA Thermometer</b><br>
 
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The flow cytometer’s data shows the highest fluorescent protein expression at the 37°C and 42°C. That proves the 37°C induced RBS (RNA thermometer) has an appropriate function.<br>
 
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The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer. <br><br>
 
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<div><img src = "http://partsregistry.org/wiki/images/4/4e/RNA_data_2.jpg" width="450"></div>
 
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<br><b>Circuit B: </b>Ptet <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> is a constitutive repressible promoter, and the RBS we use is <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. The mGFP is the gene coding for “Green Fluorescence Protein”. <br><br>
 
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<p>There are several systems suggested in literature that are based on RNA secondary structure. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded behind it (figure 1). In this way gene expression can be regulated on the RNA level by temperature.</p>
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<p>By this circuit we can realize the base line of the protein expression with common RBS and Ptet promoter. Later, we will add more parts on this circuit to regularly express target protein, so we have to build up the base line of protein expression.</p>
 
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<p>The result shows E.coli will generate better volume of protein at the 32°C and 37°C, it is a reasonable conclusion. That’s because 32-37°C is the most appropriate growth temperature for E.coli. Therefore, we get the information about the basic growth temperature and base line of protein expression in this diagram.(figure 4.)</p>
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<center><div><img src = "http://partsregistry.org/wiki/images/a/a6/RNA_Design_2.jpg"></div></center>
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<br><b>Figure 2:</b> Post-transcriptional regulation acts at the RNA level.<br>At the left , in the off-state, the RNA is folded into a hairpin structure that occludes the Shine Dalgarno region (ribosome binding site). In this situation the translation is blocked because the ribosome cannot bind to the RNA.<br>
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At the right, the temperature change causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state.  
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<div><img src = "http://partsregistry.org/wiki/images/d/d5/RNA_data_2-2.jpg" width="700"></div>
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<br><b>Figure 4: The reporter circuit:</b><br>
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The result shows E.coli will generate better volume of protein at the 32°C and 37°C, it is a reasonable conclusion. That’s because 32-37°C is the most appropriate growth temperature for E.coli. Therefore, we get the information about the basic growth temperature and base line of protein expression.<br>The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer. <br><br>
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<div><img src = "http://partsregistry.org/wiki/images/3/3b/RNA_data_3.jpg" height="85" width="860"></div>
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<br><b>Circuit A+B: </b>
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<a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> is a constitutive promoter, and the 37℃induced RBS we use is <a href="http://partsregistry.org/Part:BBa_K115002">BBa_K115002</a>.Ptet <a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a> is a constitutive repressible promoter, and the RBS we use is <a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a>. The mGFP is the gene coding for “Green Fluorescence Protein”. The terminators are all <a href="http://partsregistry.org/Part:BBa_J61048">BBa_J61048</a>.<br><br>
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<p>In this circuit, we combined the 37°C induced RBS (RNA thermometer), tetR gene and Ptet promoter with a reporter protein (GFP). We want to construct a circuit that will be turn on at low temperature and that will be turn off at high temperature. That’s why we combined 37°C induced RBS and tetR gene in the upstream circuit. When E.coli is cultivated above 37°C, it will make tetR protein to inhibit Ptet promoter and reporter protein cannot express. At the same time, E.coli will focus on expanding population and produce enough material for target product. So when we shift E.coli to lower temperature, it will smoothly produce target protein with plenty of material and no promoter inhibitor.</p>
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As the experimental data shown (figure 5.), the lower temperature 27°C and 32°C can lead E.coli to produce the greatest amount of GFP. In contrast, the higher 37°C and 42°C cause E.coli to produce GFP in slight amount.
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<div><img src = "https://static.igem.org/mediawiki/2011/f/f4/RNA_data_4.JPG" width="700"></div>
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<br><b>Figure 5: A low temperature release system with RNA thermometer</b><br>
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The lower temperature 27°C and 32°C can lead E.coli to produce the greatest amount of GFP. In contrast, the higher 37°C and 42°C cause E.coli to produce GFP in slight amount.<br>The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer. <br><br>
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<h2>Comment</h2>
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Our experimental results indicate that high temperature decreased the translation rate of the target protein, and this temperature-dependent genetic circuit can control the expression level of the target protein by the host cell's incubation temperature. However, the translational activity of the <a href = "http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002">BBa_K115002</a> at different temperatures cannot be quantified directly from experimental data. To overcome this problem, we provided a dynamic model which can quantitatively assess the translation strength of the <a href = "http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002">BBa_K115002</a> at temperatures 27°C, 32°C, 37°C and 42°C.
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</p>
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<p>We used the RNA thermometer to build up a low-temperature release system which was designed to control target protein expression. A specific ribosome binding site (RBS) <a href = "http://partsregistry.org/wiki/index.php?title=Part:BBa_K115002">BBa_K115002</a> with high translation activity at high temperature(> 37°C) and low translation activity at room temperature was used to design the temperature-dependent genetic circuit in E. coli, with a green fluorescent protein (GFP) used as the reporter protein. We analyzed fluorescence intensity during E. coli growth at log phase and stationary phase at temperatures 25°C, 30°C, 30°C and 40°C. </p>
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<p>We also gather the data of green fluorescent protein’s expression via flow cytometer.</p>
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Revision as of 13:32, 3 October 2011



RNA Thermometer

Design

RNA Thermometer system is about an RNA-based system that is able to have differential gene expression when temperature changes. The expression of heat-shock, cold-shock and some virulence genes are coordinated in response to temperature changes. Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is a widely used regulatory strategy. RNA thermometers are thermo-sensors that regulate gene expression by temperature-induced changes in RNA conformation.


Figure 1: RNA thermometer
At the left, the RNA forms stable base-pairs on the Shine-Dalgarno sequence (SD sequence), disabling the ribosome to bind. (SD sequence is the polypurine sequence AGGAGG centered about 10 bp before the AUG initiation codon on bacterial mRNA.) It is now in the on-state. The base-pairing of this RNA region will block the expression of the protein encoded behind it. In the other hand, at the right, certain external factor is added and causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state.

There are several systems suggested in literature that are based on RNA secondary structure. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded behind it (figure 1). In this way gene expression can be regulated on the RNA level by temperature.


Figure 2: Post-transcriptional regulation acts at the RNA level.
At the left , in the off-state, the RNA is folded into a hairpin structure that occludes the Shine Dalgarno region (ribosome binding site). In this situation the translation is blocked because the ribosome cannot bind to the RNA.
At the right, the temperature change causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state.

We used the RNA thermometer to build up a low-temperature release system which was designed to control target protein expression. A specific ribosome binding site (RBS) BBa_K115002 with high translation activity at high temperature(> 37°C) and low translation activity at room temperature was used to design the temperature-dependent genetic circuit in E. coli, with a green fluorescent protein (GFP) used as the reporter protein. We analyzed fluorescence intensity during E. coli growth at log phase and stationary phase at temperatures 25°C, 30°C, 30°C and 40°C.

We also gather the data of green fluorescent protein’s expression via flow cytometer.

Retrieved from "http://2011.igem.org/Team:NCTU_Formosa/RNA_data"