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

 
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<div class= "project_superrow">
 
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<div id= "page_title"><h1>Signal Co-expression</h1>
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<div id= "page_title"><h1>Parallel Sensors</h1>
 
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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">
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>P<sub>yeaR</sub></i>) </p></a>
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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">
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<p style="color:#5570b0; font-size: 130%"> <i>P<sub>araBAD</sub></i> Characterization </p></a>
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<div class="project_content">
 
<div class="project_content">
 
<div class="project_row">
 
<div class="project_row">
<h1>Expression platform</h1>
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<h1>Expression Platform</h1>
<p>To examine the possible effects of co-expression, comparison between dose dependent fluorescence response expressed by one construct in a single plasmid and two constructs in one plasmid was conducted . The purpose of the comparison relates to the design of nitrate, potassium ion and phosphate ion (NPK) sensor, which aims to combine three constructs characterised by different outputs within one plasmid. <br><br>As the substitute of the NPK inducible promoters, well-characterised promoters araBADp and lacZp were used as their biological mechanisms are better understood.  
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<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.
  
 
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</p>
<div class="project_image">
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                                    <div class="project_image">
<img src="https://static.igem.org/mediawiki/2015/9/92/Team_HKUST-Rice_2015_Arabinose_1.PNG" alt="image caption">
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<img src="https://static.igem.org/mediawiki/2015/c/ce/Team_HKUST-Rice_2015_Coex111.PNG" alt="image caption">
 
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<img src="https://static.igem.org/mediawiki/2015/b/b4/Team_HKUST-Rice_2015_Arabinose_v2.PNG" alt="image caption">
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<div class="des">
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<p style="font-size:110%"><strong>Figure 1. Brief diagrams of single and double constructs.</strong></p></div>
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<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>
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            <div class="project_image">
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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">
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<div class="des">
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<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>
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<div class="project_image">
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<img style="width:80%" src="https://static.igem.org/mediawiki/2015/7/70/Team_HKUST-Rice_2015_parasen2b.PNG" alt="image caption">
 
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<p>The construction of a double construct araBADp-lacZp allowed for a comparison between GFPmut3b and RFP measurements while changing concentrations of inducers. Two graphs of fluorescence changes according to different inducers concentrations for both single and double constructs could be analysed and compared. </p>
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<div class="des">
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<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>
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<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>
 
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<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
<h1>Method</h1>
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<h1>Constructs</h1>
                                        <p style="font-size:200%"><u> Cloning </u></p>
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<p>Constructs were built by digestion and ligation method. </p>
<p>There were two inducible single construct for characterization. They were araBADp with GFPmut3b and lacZp with mRFP induced by arabinose and IPTG respectively. Both of the constructs were built by digestion and ligation method following the RFC10 standard. </p>
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<div class="project_image">
 
<div class="project_image">
<img src="https://static.igem.org/mediawiki/2015/d/d3/Team_HKUST-Rice_2015_Arabinose_3.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>
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<div class="des">
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<p style="font-size:110%"><strong>Figure 3a. Inducible single constructs <i>P<sub>araBAD</sub></i> - <i>gfpmut3b</i>.</strong></p></div>
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<div class="project_image">
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<img style="width:60%;" src="https://static.igem.org/mediawiki/2015/9/9e/Team_HKUST-Rice_2015_Coex3b.PNG" alt="image caption"></div>
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<div class="des">
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<p style="font-size:110%"><strong>Figure 3b. Inducible single constructs <i>P<sub>lac</sub></i>- <i>mrfp</i>.</strong></p></div>
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<div class="project_image">
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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">
 
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</div>
<p>Same approach was also adopted for the double construct to ligate araBADp and lacZp together.</p>  
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<div class="des">
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<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>
 
 
 
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<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
<h1>Experiment that we did</h1>
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<h1>Experiments Performed</h1>
                                        <p style="font-size:200%"><u> Characterization </u></p>
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<p>As all of them were inducible constructs, fluorescence would only be expressed when inducers were present.
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<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>For every single characterization, same procedures were followed. First, the sample which would be measured had to be inoculated in Falcon tubes. In the next day, 25-fold dilution was carried out for the inoculated samples by using the M9 minimal medium with specific inducers and concentrations. They would then be transferred into 96-well deep well plate for overnight induction. Again, the sample would be further diluted by ten-fold dilution in the next day. Several hours were needed to let the cells grow from lag phase to log phase. The OD600 should be around 0.4-0.7 ideally and kept constant for every trial. Ultimately, the result could be gathered with the help of EnVision® Multilabel Reader(OD595) using filter 485/14nm FITC and 535/25nm FITC for excitation and emission measurement respectively. All data would be plotted as graphs for further analysis.  
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<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>
<p style="font-size:200%"><u> Preparing medium concentration  </u></p>
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<p>M9 minimal medium was used for inoculation, making medium with inducers and dilution. It was chosen because of its low auto-fluorescence. Serial dilution was usually adopted for making medium with different concentration of inducers.
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Experiments performed
</P>
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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.
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</div>
 
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<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
<h1>Result obtained</h1>
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<h1 id="co-expression">Results</h1>
<p>After obtaining characterization data for the araBADp-GFPmut3b single construct, the dose response curve was compared with that expressed in a double construct (with lacZp-RFP) .
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<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 src="https://static.igem.org/mediawiki/2015/e/ec/Team_HKUST-Rice_2015_CE_Result_1.PNG" alt="image caption">
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<img style="width:90%;" src="https://static.igem.org/mediawiki/2015/b/b1/Team_HKUST-Rice_2015_para42.PNG" alt="image caption" >
 
</div>
 
</div>
<p>From figure 5a, it can be observed that in a double construct, the maximum expression of GFPmut3b decreases for the arabinose inducible araBADp system. Furthermore, from figure 5b, it is shown that when the coexpressed system is under full induction, the curve for the double construct araBADp shifts to the right compared to both the single construct and double construct without induction.
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<div class="des">
<br><br>In order to further investigate the effect of induction level of the coexpressed lacZp-RFP construct on the GFPmut3b expression of the double construct araBADp system, experiments were carried out varying the IPTG induction concentrations. </p>
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<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>
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 +
<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 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>
 
<div class="project_image">
 
<div class="project_image">
<img src="https://static.igem.org/mediawiki/2015/0/01/Team_HKUST-Rice_2015_CE_Result_2.PNG" alt="image caption">
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<img src="https://static.igem.org/mediawiki/2015/7/7f/Team_HKUST-Rice_2015_Coex6.PNG" alt="image caption">
 
</div>
 
</div>
 
 
<p>Figure 6 exhibit that varying the induction level of the coexpressed lacZp-RFP construct seems to have an effect on the expression level of araBADp-GFPmut3b in a double construct. At a higher IPTG concentration, the maximum expression level decreases. However, no trend can be observed for the horizontal shift of the curve, though at the highest IPTG concentration the graph observed to shift right, no significant shift at lower IPTG concentrations.</p>
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<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.  
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</p>
 
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                               <div class="project_row">
 
                               <div class="project_row">
 
<hr class="para">
 
<hr class="para">
 
<h1>Conclusion</h1>
 
<h1>Conclusion</h1>
                               <p>By using the araBADp-GFPmut3b and lacZp-RFP 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 decreased compared to the single construct induced araBADp-GFPmut3b system whether the coexpressed construc
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                               <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>
t was induced or not. We conjecture that it is possibly due to the limited cellular resources(eg. aminoacids, enzymes required for gene translation/transcription) and the synthesised proteins (eg.lacI, RFP) that could increase the cell load and affects its growth. However, since the investigation was done only focusing on one combination of inducible constructs(araBADp-GFPmut3b and lacZp-RFP) it may not be used to generalise the effect of expressing a charactered construct in a double construct, including that for the N,P and K construct. Thus further improvements should be made.</p>
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                                 <div class="project_row">
 
                                 <div class="project_row">
 
<hr class="para">
 
<hr class="para">
 
<h1>Further Improvements</h1>
 
<h1>Further Improvements</h1>
                                      <p style="font-size:200%"><u> Progress made in brief: </u></p>
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<p><b>araBADp-GFPmut3b and lacZp-RFP characterisation</b>
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<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>Measuring the RFU (GFPmut3b and RFP respectively) of the of E.Coli  samples, in increasing concentrations of respective inducers (for both Arabinose and IPTG), using 3 biological replicates and 2 technical replicates.
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<br><br><b>araBADp GFPmut3b-lacZp RFP characterisation</b>
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            <br>Measuring the RFU (GFPmut3b and RFP respectively) of the of E.Coli  samples, in increasing concentrations of inducers, both Arabinose and IPTG, using 3 biological replicates and 2 technical replicates.</p>
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                              <p style="font-size:200%"><u> Improvements: </u></p>
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<p>Repeat number of experiments and biological and technical replicates until data is reliable and accurate.
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          <br>With reliable and accurate data, conclusion made from this experiment can be used for  further investigation.
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<br><br> Increase number of concentrations of inducers used within the same range to obtain detailed data from our experiments.
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<br><br>Characterize Plux GFPmut3b and PLux GFPmut3b- lacZp RFP.
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          <BR>Measuring the RFU (GFPmut3b and RFP respectively) of the of E.Coli  samples, in increasing concentrations of inducers, HSL and IPTG respectively, and HSL and IPTG respectively.</p>
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<p style="font-size:200%"><u> Summary of two promoter system in one construct we want to work on: </u></p>
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<p><b>Charaterize araBADp-GFPmut3b lacZp-RFP with a third promoter system (i.e. PTet and PLux)</b>
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<br>This will give a more reliable comparison of more than one promoter system, as opposed to one system.
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<br><br><b>Change order of the three promoter system and characterize the each construct, by measuring their RFU in increasing concentration of the three inducers  Arabinose, IPTG and ATC)</b>
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<br>After comparing results of the three types of promoter system within each other, we can apply the data to the projects’ actual sensors and make a hypothesis of if combining the three sensors will affect the actual results.
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<br><br>Furthermore, we can start on characterizing the threes sensors in one promoter construct, 2 promoter construct, and three promoter construct and compare their results. We can do this by using similar methods as used above.
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<br><br>Compare two promoter system as opposed to one, using the Nitrogen, Phosphate, and Potassium sensors by characterizing them with their respective inducers.
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+
<br><br>Compare three promoter system as opposed to two and one using the Nitrogen, Phosphate, and Potassium sensors by characterizing them with their respective inducers.
+
  
<br><br>
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<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 NPK promoter system, as   
 +
          opposed to a double system.
  
</p>
+
<br><br>Co-express potassium, phosphate and nitrate sensors and their respective inducers.
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</p></div>
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                                        <hr class="para">
 +
<h2>References</h2>
 +
                                <p style="font-size:125%">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.
 +
<br><br>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.
 +
<br><br>Wang, B., Barahona, M., & Buck, M. (2015). Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities. Nucleic acids research, gku1388.
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</p></div>
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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.

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Figure 3a. Inducible single constructs ParaBAD - gfpmut3b.

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Figure 3b. Inducible single constructs Plac- mrfp.

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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.

Wang, B., Barahona, M., & Buck, M. (2015). Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities. Nucleic acids research, gku1388.