Difference between revisions of "Team:HKUST-Rice/Expression"

 
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<div id="MYicon1">
 
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                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"><img src="https://static.igem.org/mediawiki/2015/e/ea/HKUST-Rice15_leftarrow.png">
 
                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"><img src="https://static.igem.org/mediawiki/2015/e/ea/HKUST-Rice15_leftarrow.png">
<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (P<sub>yeaR</sub>) </p></a>
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>P<sub>yeaR</sub></i>) </p></a>
 
</div>
 
</div>
  
 
                 <div id="MYicon2">
 
                 <div id="MYicon2">
                       <a href="https://2015.igem.org/Team:HKUST-Rice/TXTL"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
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                       <a href="https://2015.igem.org/Team:HKUST-Rice/Expression/ParaBAD"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
<p style="color:#5570b0; font-size: 130%"> TXTL </p></a>
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<p style="color:#5570b0; font-size: 130%"> <i>P<sub>araBAD</sub></i> Characterization </p></a>
 
</div>
 
</div>
  
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<div class="project_content">
 
<div class="project_content">
 
<div class="project_row">
 
<div class="project_row">
<h1>Expression platform</h1>
+
<h1>Expression Platform</h1>
<p>To examine the possible effects of co-expression, the dose-dependent fluorescence response of one construct in a single plasmid was compared to that of two constructs in a single plasmid. The purpose of the comparison relates to the design of our potassium, phosphate and nitrate (KPN) sensor, which aims to combine three constructs characterized by different outputs within a single plasmid.
+
<p>To examine the possible effects of parallel sensing, the dose-dependent fluorescence response of one construct in a single plasmid was compared to that of two constructs in a single plasmid. The purpose of the comparison relates to the design of our potassium, phosphate and nitrate (NPK) sensor, which aims to combine three constructs characterized by different outputs within a single plasmid.
  
 
</p>
 
</p>
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<div class="des">
 
<div class="des">
 
<p style="font-size:110%"><strong>Figure 1. Brief diagrams of single and double constructs.</strong></p></div>
 
<p style="font-size:110%"><strong>Figure 1. Brief diagrams of single and double constructs.</strong></p></div>
<p>The construction of a double construct P<sub>araBAD</sub>-P<sub>lacZ</sub> allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of the individual inducers. Graphs of changes in fluorescence according to different inducer concentrations for both single and double constructs could be analysed and compared. </p>
+
<p>As substitutes for the NPK-inducible promoters, <i>P<sub>araBAD</sub></i> and <i>P<sub>lac</sub></i> were used since their biological mechanisms are understood better. </p>
 
            <div class="project_image">
 
            <div class="project_image">
<img style="width:80%" src="https://static.igem.org/mediawiki/2015/1/1c/Team_HKUST-Rice_2015_araCfinal.PNG" alt="image caption">
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<img style="width:80%" src="https://static.igem.org/mediawiki/2015/c/c8/Team_HKUST-Rice_2015_parasen2a.PNG" alt="image caption">
 
</div>
 
</div>
 
<div class="des">
 
<div class="des">
<p style="font-size:110%"><strong>Figure 2a. Schematic diagram of the inducible P<sub>araBAD</sub>-GFP construct.</strong> In the absence of L-Arabinose, AraC is constitutively produced, repressing P<sub>araBAD</sub>. In the presence of L-Arabinose, AraC protein driven by the constitutive promoter binds with L-Arabinose and induces P<sub>araBAD</sub>, triggering the production of GFP.</p></div>
+
<p style="font-size:110%"><strong>Figure 2a. Schematic diagram of the inducible <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> construct.</strong> In the absence of L-Arabinose, AraC is constitutively produced, repressing <i>P<sub>araBAD</sub></i>. In the presence of L-Arabinose, AraC protein driven by the constitutive promoter binds with L-Arabinose and induces <i>P<sub>araBAD</sub></i>, triggering the production of GFPmut3b.</p></div>
 
 
 
<div class="project_image">
 
<div class="project_image">
<img style="width:80%" src="https://static.igem.org/mediawiki/2015/1/17/Team_HKUST-Rice_2015_lacfinal.PNG" alt="image caption">
+
<img style="width:80%" src="https://static.igem.org/mediawiki/2015/7/70/Team_HKUST-Rice_2015_parasen2b.PNG" alt="image caption">
</div>\
+
</div>
 
<div class="des">
 
<div class="des">
<p style="font-size:110%"><strong>Figure 2b. Schematic diagram of the inducible P<sub>lacZ</sub>-mRFP construct.</strong> In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses P<sub>lacZ</sub>. When IPTG is present, LacI protein is repressed, triggering the production of mRFP.</p></div>
+
<p style="font-size:110%"><strong>Figure 2b. Schematic diagram of the inducible <i>P<sub>lac</sub></i> - <i>mrfp</i> construct.</strong> In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses <i>P<sub>lac</sub></i>. When IPTG is present, LacI protein is repressed, triggering the production of mRFP.</p></div>
<p>The construction of a double construct P<sub>araBAD</sub>-GFPmut3b-P<sub>lacZ</sub>-mRFP allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of inducers. Dose response curve for both single and double constructs could then be analysed and compared. </p>
+
<p>The construction of a double construct <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> - <i>P<sub>lac</sub></i> - <i>mrfp</i> allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of inducers. Dose response curves for both single and double constructs could then be analyzed and compared. </p>
 
</div>
 
</div>
 
<div class="project_row">
 
<div class="project_row">
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<p>Constructs were built by digestion and ligation method. </p>
 
<p>Constructs were built by digestion and ligation method. </p>
 
<div class="project_image">
 
<div class="project_image">
<img style="width:100%;" src="https://static.igem.org/mediawiki/2015/b/bc/Team_HKUST-Rice_2015_3a3b.PNG" alt="image caption">
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<img style="width:60%;" src="https://static.igem.org/mediawiki/2015/4/4e/Team_HKUST-Rice_2015_Coex3a.PNG" alt="image caption"></div>
</div>
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<div class="des">
 
<div class="des">
<p style="font-size:110%"><strong>Figure 3. Inducible double construct P<sub>araBAD</sub>-GFP-P<sub>lacZ</sub>-mRFP.</strong>.</p></div>
+
<p style="font-size:110%"><strong>Figure 3a. Inducible single constructs <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i>.</strong></p></div>
 +
<div class="project_image">
 +
<img style="width:60%;" src="https://static.igem.org/mediawiki/2015/9/9e/Team_HKUST-Rice_2015_Coex3b.PNG" alt="image caption"></div>
 +
<div class="des">
 +
<p style="font-size:110%"><strong>Figure 3b. Inducible single constructs <i>P<sub>lac</sub></i>- <i>mrfp</i>.</strong></p></div>
  
 
<div class="project_image">
 
<div class="project_image">
<img style="width:80%;" src="https://static.igem.org/mediawiki/2015/e/ea/Team_HKUST-Rice_2015_fig4.PNG" alt="image caption">
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<img style="width:80%;" src="https://static.igem.org/mediawiki/2015/0/02/Team_HKUST-Rice_2015_Coex4.PNG" alt="image caption">
 
</div>
 
</div>
 
<div class="des">
 
<div class="des">
<p style="font-size:110%"><strong>Figure 4. Inducible construct (a) P<sub>araBAD</sub>-GFP and (b) P<sub>lacZ</sub>-mRFP.</strong>.</p></div>
+
<p style="font-size:110%"><strong>Figure 4. Inducible double construct <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> - <i>P<sub>lac</sub></i> - <i>mrfp</i>.</strong></p></div>
 
 
 
</div>
 
</div>
 
<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
<h1>Experiments performed</h1>
+
<h1>Experiments Performed</h1>
 
                      
 
                      
<p>For all experiments, the bacteria were first grown overnight, then a 25-fold dilution was carried out using M9 minimal medium with specific inducers and concentrations. After that, they were transferred into a 96-well deep well plate for overnight induction. The samples were then further diluted ten-fold the next day. The cells were allowed to grow from lag phase to log phase for several hours. Ultimately, the result was gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis.  
+
<p>For all experiments, the bacteria were first grown overnight and diluted 25-fold in M9 minimal media with specific inducers and concentrations. After that, the dilutions were transferred to a 96-well deep well plate for overnight induction. The samples were then diluted 10-fold the next day and were allowed to grow from lag phase to log phase for several hours. Ultimately, the measurements were gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis.  
<br><br>Please visit <a href="https://static.igem.org/mediawiki/2015/a/a7/Team_HKUST-Rice_2015_Signal_Co.pdf"target="_blank">Parallel Sensors Experiment Protocol</a> for more details.
+
<br><br>Please visit <a href="https://static.igem.org/mediawiki/2015/e/eb/Team_HKUST-Rice_2015_2_parallelvvvvvvv.pdf"target="_blank">Parallel Sensor Experiment Protocol</a> for more details.
 
</p>
 
</p>
 +
 +
Experiments performed
 +
For all experiments, the bacteria were grown overnight and were diluted 25-fold in M9 minimal medium with appropriate inducer concentrations. After that, they were transferred into a 96-well deep well plate for overnight growth. The samples were further diluted ten-fold the next day and allowed to grow to log phase. Ultimately, fluorescence data was gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis.
 +
 +
Please visit Parallel Sensors Experiment Protocol for more details.
  
 
</div>
 
</div>
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<hr class="para">
 
<hr class="para">
 
<h1 id="co-expression">Results</h1>
 
<h1 id="co-expression">Results</h1>
<p >After obtaining characterization data for the P<sub>araBAD</sub>-GFPmut3b single construct, the dose response curve was compared with that expressed in a double construct (with P<sub>lacZ</sub>-mRFP).
+
<p >After obtaining characterization data for the <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> single construct, the dose response curve was compared with the graph of the double construct (with <i>P<sub>lac</sub></i> - <i>mrfp</i>).
 
</p>
 
</p>
 
<div class="project_image">
 
<div class="project_image">
<img style="width:90%;" src="https://static.igem.org/mediawiki/2015/3/37/Team_HKUST-Rice_2015_fig5.PNG" alt="image caption" >
+
<img style="width:90%;" src="https://static.igem.org/mediawiki/2015/b/b1/Team_HKUST-Rice_2015_para42.PNG" alt="image caption" >
 
</div>
 
</div>
 
<div class="des">
 
<div class="des">
<p style="font-size:110%"><strong>Figure 5 . Response curve of P<sub>araBAD</sub>-GFP in single construct compared with in double construct plasmid (a) Co-expressed construct (P<sub>lacZ</sub>-mRFP) with full induction (5mM IPTG) (b) co-expressed construct without induction</strong>.</p></div>
+
<p style="font-size:110%"><strong>Figure 5 . Response curve of <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> in single construct compared to the double construct plasmid</strong> (a) Constructs used in the experiment (b) Co-expressed construct (<i>P<sub>lac</sub></i> - <i>mrfp</i>) with full induction (5 mM IPTG) (c) co-expressed construct without induction</strong>.</p></div>
 
 
<p>From Figure 5a, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible P<sub>araBAD</sub> system. Furthermore, from Figure 5b, it is shown that when the co-expressed system is under full induction, the curve for the double construct P<sub>araBAD</sub> shifts to the right compared to both the single construct and double construct without induction, indicating a decrease in sensitivity.
+
<p>From Figure 5b, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible <i>P<sub>araBAD</sub></i> system. Furthermore, Figure 5c shows that the curve for the double construct <i>P<sub>araBAD</sub></i> shifts to the right when the co-expressed system is under full induction, indicating a decrease in sensitivity.
 
   
 
   
<br><br>In order to further investigate the effect of induction level of the co-expressed P<sub>lacZ</sub>-mRFP construct on the GFPmut3b expression of the double construct P<sub>araBAD</sub> system, experiments were carried out using varying IPTG concentrations.  
+
<br><br>In order to investigate the effect of induction levels of <i>P<sub>lac</sub></i> - <i>mrfp</i> on the GFPmut3b expression in the double construct <i>P<sub>araBAD</sub></i> system, experiments were carried out using varying IPTG concentrations.  
 
  </p>
 
  </p>
 
<div class="project_image">
 
<div class="project_image">
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</div>
 
</div>
 
 
<p>Figure 6 exhibits that varying the induction level of the co-expressed P<sub>lacZ</sub>-mRFP construct seems to have an effect on the expression level of P<sub>araBAD</sub>-GFPmut3b in a double construct. At a higher IPTG concentration, the maximum expression of GFPmut3b decreases. However, no trend can be observed for the horizontal shift of the curve, except at the highest IPTG concentration (2<sup>2</sup> mM) where the graph shifts to the right. No significant shift was observed at lower IPTG concentrations.  
+
<p>Figure 6 shows that variations in the induction level of the co-expressed <i>P<sub>lac</sub></i> - <i>mrfp</i> construct affects the expression level of <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> in a double construct. At a higher IPTG concentration, the maximum expression of GFPmut3b decreases. However, no trend is observed for the horizontal shift of the curve except at the highest IPTG concentration (2<sup>2</sup> mM), where the graph shifts to the right. No significant shift was observed at lower IPTG concentrations.  
 
</p>
 
</p>
 
</div>
 
</div>
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<hr class="para">
 
<hr class="para">
 
<h1>Conclusion</h1>
 
<h1>Conclusion</h1>
                               <p>By using the P<sub>araBAD</sub>-GFPmut3b and P<sub>lacZ</sub>-mRFP inducible systems, the possible difference of dose dependent fluorescence expression in a double construct and single construct was investigated. In a double construct, the maximal expression was lower than the single construct induced P<sub>araBAD</sub>-GFPmut3b, whether or not the co-expressed construct was induced. We conjecture that it is possibly due to the limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription etc.) and the synthesized proteins (e.g. lacI, mRFP) that could increase the cell load and affect its growth. However, since the investigation was done focusing only on one combination of inducible constructs(P<sub>araBAD</sub>-GFPmut3b and P<sub>lacZ</sub>-mRFP), the data obtained cannot be assumed to apply to all instances of expressing a charactered construct in a double construct, including that for the KPN construct. Thus, further improvements should be made.</p>
+
                               <p>By using the <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> and <i>P<sub>lac</sub></i> -<i>mrfp</i> inducible systems, the possible difference of dose-dependent fluorescence expression in a double construct and single construct was investigated. In a double construct, the maximal expression was lower than the single construct induced <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i>, whether or not the co-expressed construct was induced. We hypothesize that this is due to limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription etc.) since the synthesized proteins (e.g. lacI, mRFP) could increase the cell load and affect its growth. However, since the investigation focused on only one combination of inducible constructs (<i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i> and <i>P<sub>lac</sub></i> - <i>mrfp</i>), the data obtained cannot be generalized to all instances related to expression of a characterized construct in a double construct, including those of the NPK constructs. Thus, further improvements should be made.</p>
 
</div>
 
</div>
 
                                 <div class="project_row">
 
                                 <div class="project_row">
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<h1>Further Improvements</h1>
 
<h1>Further Improvements</h1>
 
                                                                
 
                                                                
<p>Since characterization data from one combination of double construct cannot be applied to all double constructs in general, characterization of more combinations of inducible reporter systems (eg. P<sub>luxR</sub>-GFPmut3-P<sub>lacZ</sub>-mRFP, P<sub>tetO</sub>-GFPmut3b-P<sub>lacZ</sub>-mRFP) is required.  
+
<p>Since characterization data from one combination of double constructs cannot be applied to all double constructs in general, characterization of more combinations of inducible reporter systems (eg. <i>P<sub>luxR</sub></i> - <i>gfpmut3b</i> - <i>P<sub>lac</sub></i> - <i>mrfp</i>, <i>P<sub>tetO</sub></i> - <i>gfpmut3b</i> - <i>P<sub>lac</sub></i> - <i>mrfp</i>) is required.  
  
<br><br>Charaterize P<sub>araBAD</sub>-GFPmut3b P<sub>lacZ</sub>-mRFP with a third construct (i.e. P<sub>tetO</sub> and P<sub>luxR</sub>)
+
<br><br>Characterize <i>P<sub>BAD</sub></i> - <i>gfpmut3b</i>- <i>P<sub>lac</sub></i> - <i>mrfp</i> with a third construct (i.e. <i>P<sub>tetO</sub></i> and <i>P<sub>luxR</sub></i>)
           This will give a more reliable comparison for a triple construct KPN promoter system, as     
+
           This will give a more reliable comparison for a triple construct NPK promoter system, as     
 
           opposed to a double system.
 
           opposed to a double system.
  
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{{HKUST-Rice Directory}}

Latest revision as of 17:22, 18 September 2015


Parallel Sensors

Expression Platform

To examine the possible effects of parallel sensing, the dose-dependent fluorescence response of one construct in a single plasmid was compared to that of two constructs in a single plasmid. The purpose of the comparison relates to the design of our potassium, phosphate and nitrate (NPK) sensor, which aims to combine three constructs characterized by different outputs within a single plasmid.

image caption

Figure 1. Brief diagrams of single and double constructs.

As substitutes for the NPK-inducible promoters, ParaBAD and Plac were used since their biological mechanisms are understood better.

image caption

Figure 2a. Schematic diagram of the inducible ParaBAD - gfpmut3b construct. In the absence of L-Arabinose, AraC is constitutively produced, repressing ParaBAD. In the presence of L-Arabinose, AraC protein driven by the constitutive promoter binds with L-Arabinose and induces ParaBAD, triggering the production of GFPmut3b.

image caption

Figure 2b. Schematic diagram of the inducible Plac - mrfp construct. In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses Plac. When IPTG is present, LacI protein is repressed, triggering the production of mRFP.

The construction of a double construct ParaBAD - gfpmut3b - Plac - mrfp allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of inducers. Dose response curves for both single and double constructs could then be analyzed and compared.


Constructs

Constructs were built by digestion and ligation method.

image caption

Figure 3a. Inducible single constructs ParaBAD - gfpmut3b.

image caption

Figure 3b. Inducible single constructs Plac- mrfp.

image caption

Figure 4. Inducible double construct ParaBAD - gfpmut3b - Plac - mrfp.


Experiments Performed

For all experiments, the bacteria were first grown overnight and diluted 25-fold in M9 minimal media with specific inducers and concentrations. After that, the dilutions were transferred to a 96-well deep well plate for overnight induction. The samples were then diluted 10-fold the next day and were allowed to grow from lag phase to log phase for several hours. Ultimately, the measurements were gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis.

Please visit Parallel Sensor Experiment Protocol for more details.

Experiments performed For all experiments, the bacteria were grown overnight and were diluted 25-fold in M9 minimal medium with appropriate inducer concentrations. After that, they were transferred into a 96-well deep well plate for overnight growth. The samples were further diluted ten-fold the next day and allowed to grow to log phase. Ultimately, fluorescence data was gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis. Please visit Parallel Sensors Experiment Protocol for more details.

Results

After obtaining characterization data for the ParaBAD - gfpmut3b single construct, the dose response curve was compared with the graph of the double construct (with Plac - mrfp).

image caption

Figure 5 . Response curve of ParaBAD - gfpmut3b in single construct compared to the double construct plasmid (a) Constructs used in the experiment (b) Co-expressed construct (Plac - mrfp) with full induction (5 mM IPTG) (c) co-expressed construct without induction.

From Figure 5b, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible ParaBAD system. Furthermore, Figure 5c shows that the curve for the double construct ParaBAD shifts to the right when the co-expressed system is under full induction, indicating a decrease in sensitivity.

In order to investigate the effect of induction levels of Plac - mrfp on the GFPmut3b expression in the double construct ParaBAD system, experiments were carried out using varying IPTG concentrations.

image caption

Figure 6 shows that variations in the induction level of the co-expressed Plac - mrfp construct affects the expression level of ParaBAD - gfpmut3b in a double construct. At a higher IPTG concentration, the maximum expression of GFPmut3b decreases. However, no trend is observed for the horizontal shift of the curve except at the highest IPTG concentration (22 mM), where the graph shifts to the right. No significant shift was observed at lower IPTG concentrations.


Conclusion

By using the ParaBAD - gfpmut3b and Plac -mrfp inducible systems, the possible difference of dose-dependent fluorescence expression in a double construct and single construct was investigated. In a double construct, the maximal expression was lower than the single construct induced ParaBAD - gfpmut3b, whether or not the co-expressed construct was induced. We hypothesize that this is due to limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription etc.) since the synthesized proteins (e.g. lacI, mRFP) could increase the cell load and affect its growth. However, since the investigation focused on only one combination of inducible constructs (ParaBAD - gfpmut3b and Plac - mrfp), the data obtained cannot be generalized to all instances related to expression of a characterized construct in a double construct, including those of the NPK constructs. Thus, further improvements should be made.


Further Improvements

Since characterization data from one combination of double constructs cannot be applied to all double constructs in general, characterization of more combinations of inducible reporter systems (eg. PluxR - gfpmut3b - Plac - mrfp, PtetO - gfpmut3b - Plac - mrfp) is required.

Characterize PBAD - gfpmut3b- Plac - mrfp with a third construct (i.e. PtetO and PluxR) This will give a more reliable comparison for a triple construct NPK promoter system, as opposed to a double system.

Co-express potassium, phosphate and nitrate sensors and their respective inducers.


References

Ang, J., Harris, E., Hussey, B. J., Kil, R., & McMillen, D. R. (2013). Tuning response curves for synthetic biology. ACS synthetic biology, 2(10), 547-567.

Wang, B., Barahona, M., & Buck, M. (2013). A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals. Biosensors and Bioelectronics, 40(1), 368-376.

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