Difference between revisions of "Team:BIT-China/Proof"

 
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                 <!--问题描述-->
 
                 <!--问题描述-->
 
                 <div class="problem-txt block-content col-sm-12" style="margin-top: 10px">
 
                 <div class="problem-txt block-content col-sm-12" style="margin-top: 10px">
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                    <div class="problem-title">Proof of concept</div>
 
 
                     <div>
 
                     <div>
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                         <span>
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                         To prove our concept that:
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                            To prove our concept that we can make the bacteria sense the plasmid numbers and keep the numbers above a threshold, we separately designed two parts. One is to prove that plasmid numbers will influence the inhibitor concentration, the other is to prove the inhibitor concentration can adjust the expression of killer gene.
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                    </div>
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                            <a href="https://2016.igem.org/Team:BIT-China/Results">See our final results here!</a>
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                    <div class="block-paragraph">
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                        </span>
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                        (1) We can make the bacteria sense the plasmid numbers.
 +
                        <br>(2) The in-promoter will respond differently to different signal which
 +
                        can reflect the plasmids losing on different levels. This way, our system can
 +
                        control the plasmids numbers above a threshold.
 +
                    </div>
 +
                    <div class="block-paragraph">
 +
                        We designed two parts. One of them is to prove that plasmid numbers will influence the inhibitor concentration. The other one is to prove the inhibitor concentration can regulate the expression of the killer gene by affecting its in-promoter.
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                     <div class="block-content-item">
 
                     <div class="block-content-item">
 
                         <div class="block-content-item-block">
 
                         <div class="block-content-item-block">
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                             <div style="color:#923F91;" id="#influence">
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                                 <i class="fa fa-magic" aria-hidden="true"></i>
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                             <div style="color:#923F91;margin-top: 20px" id="influence">
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                                <span class="block-content-header">
+
                                  
 +
                                <span class="block-content-header" style="text-align:left">
 +
<i class="fa fa-magic" aria-hidden="true"></i>
 
                                     Plasmid numbers will influence the inhibitor concentration
 
                                     Plasmid numbers will influence the inhibitor concentration
 
                                 </span>
 
                                 </span>
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 We use plasmids with different copies to adjust the plasmid numbers. To better simulate the real situation for application, we employed the constitutive promoter.
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                                 Because of the difficulty of controlling the number of plasmids, we can only choose some typical copy numbers of plasmids in our system.
 +
                                <br>The copy numbers are shown in the table below:
 +
                            </div>
 +
                            <div class="pic_info"><b>Table.1</b> The copy numbers of different plasmids.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 We built devices containing constitutive promoters with different strengths.
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                                 <img src="https://static.igem.org/mediawiki/2016/9/9f/T--BIT-China--Project--Proof--table1.png"
 +
                                    alt="table1" class="center-block" style="width: 300px;">
 
                             </div>
 
                             </div>
 +
                            <div class="block-paragraph">
 +
                                We also constructed the gene circuits containing constitutive promoters with
 +
                                different strengths to express the inhibitor. By this we regulate the threshold
 +
                                of plasmid numbers to meet different needs.
 +
                            </div>
 +
 
                             <div>
 
                             <div>
 
                                 <img src="https://static.igem.org/mediawiki/2016/b/bf/T--BIT-China--Project--Proof--fig1.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/b/bf/T--BIT-China--Project--Proof--fig1.png"
 
                                     alt="fig1" class="center-block" style="width: 60%;">
 
                                     alt="fig1" class="center-block" style="width: 60%;">
 
                             </div>
 
                             </div>
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                            <div>
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                          <div class="pic_info"><b>Fig.1</b> The device constructed to express the inhibitor with a constitutive promoter. </div>
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                                Meanwhile, we <b style="color: #BD6C13;">use RFP to replace the inhibitor gene</b> to indicate the inhibitor concentration.
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                            <div class="block-paragraph">
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                                 We have chosen plasmids with different copies and separately ligated these circuits on the vectors.
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                                 There are four promoters with different strengths and two kinds of RBS we have chosen:
 
                             </div>
 
                             </div>
 +
                          <div class="pic_info"><b>Table.2</b> The strengths and efficiencies of different promoters and RBS.</div>
 
                             <div>
 
                             <div>
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                                 <img src="https://static.igem.org/mediawiki/2016/8/82/T--BIT-China--Project--Proof--fig2.png"
+
                                 <img src="https://static.igem.org/mediawiki/2016/4/44/T--BIT-China--Project--Proof--table2.png"
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                                     alt="fig2" style="width: 40%;" class="center-block">
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                                     alt="table1" class="center-block" style="width: 90%;">
 
                             </div>
 
                             </div>
  
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                             <div>
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                             <div class="block-paragraph">
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                                 Through testing, we got data describing the relationship between the inhibitor concentration and plasmid copies.
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                                 Meanwhile, the RFP used to replace the inhibitor protein can directly represent the
 +
                                concentration of the inhibitor in the cell. We separately constructed these circuits on different vectors which have different copy numbers.
 +
 
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                At first, we use the modeling to explain the relationship of the concentration of the inhibitor and plasmids number like the curve below under a certain condition
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 <img src="" alt="曲线图暂时没有">
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                                 <img src="https://static.igem.org/mediawiki/2016/a/ae/T--BIT-China--Project--Proof--curve1.png" alt="曲线图暂时没有1" style="width:60%" class="center-block">
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                In the wet experiment, after the same amount of time, we will measure the RFP intensity to get the data which can describe the relationship between the inhibitor concentration and the plasmid copy numbers.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
−
                                 This way, we verified that plasmid numbers will influence the inhibitor concentration.
+
                                <img src="https://static.igem.org/mediawiki/2016/c/c2/T--BIT-China--Project--Results--Threshold--3table3.png" alt="曲线图暂时没有2" style="width:60%" class="center-block">
 +
 
 +
 
 +
        <div class="block-paragraph">
 +
            The strength of J23106 is stronger J23116, we can clearly know the inhibitor protein have a positive correlation with plasmids number.
 +
        </div>
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                 This way, we verified that plasmid numbers will influence the inhibitor concentration on the plasmid vector.
 
                             </div>
 
                             </div>
  
  
−
                             <div style="color:#923F91;" id="#adjust">
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                             <div style="color:#923F91;margin-top: 20px" id="adjust">
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                                <i class="fa fa-magic" aria-hidden="true"></i>
+
 
                                 <span class="block-content-header">
 
                                 <span class="block-content-header">
−
                                     Inhibitor concentration will adjust the expression of killer gene
+
                                <i class="fa fa-magic" aria-hidden="true"></i>
 +
                                     The inhibitor concentration can regulate the expression of the killer gene
 
                                 </span>
 
                                 </span>
 
                             </div>
 
                             </div>
−
 
 
                             <div>
 
                             <div>
−
                                 We use arabinose induced promoter to control the expression of inhibitor protein. This way, through adding different concentration of arabinose, we can adjust the inhibitor concentration.
+
                                 In order to know the relationship between the inhibitor and in-promoter, we use the arabinose induced promoter P<sub>BAD</sub> to express the inhibitor. So we can add arabinose with different concentrations to induce the promoter and create an environment with different concentrations of intracellular inhibitor. Killer gene is replaced by <span class="italic">RFP</span> under control of in-promoter.
 +
 
 
                             </div>
 
                             </div>
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                             <div>
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                             <div class="block-paragraph">
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                                 We built three devices containing different kinds of inhibitors.
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                                 We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 <img src="https://static.igem.org/mediawiki/2016/3/3d/T--BIT-China--Project--Proof--fig22.png"
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                                 <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
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                                     alt="fig2" style="width: 60%;" class="center-block">
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                                     alt="fig4" style="width: 60%;" class="center-block">
 
                             </div>
 
                             </div>
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                             <div>
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                          <div class="pic_info"><b>Fig.4</b> Arabinose induced expression cassette of three kinds of inhibitors.</div>
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                                 Meanwhile, we built three corresponding in-promoter circuits and used RFP to replace the killer gene.
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                             <div class="block-paragraph">
 +
                                 Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 <img src="https://static.igem.org/mediawiki/2016/0/0f/T--BIT-China--Project--Proof--fig3.png"
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                                 <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
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                                     alt="fig3" style="width: 60%;" class="center-block">
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                                     alt="fig5" style="width: 50%;" class="center-block">
 
                             </div>
 
                             </div>
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                             <div>
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                             <div class="pic_info"><b>Fig.5</b> In-promoter controlled expression cassette of <span class="italic">RFP</span>. <span class="italic">RFP</span> is used to replace killer gene.</div>
 +
                            <div class="block-paragraph">
 
                                 We assembled these corresponding circuits together for the final testing.
 
                                 We assembled these corresponding circuits together for the final testing.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
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                                 <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
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                                     alt="fig4" style="width: 60%;" class="center-block">
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                                     alt="fig6" style="width: 100%;" class="center-block">
 
                             </div>
 
                             </div>
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                             <div>
+
<div class="pic_info"><b>Fig.6</b> Assembly of inhibitor and "in-promoter". They can be used to test the minimum arabinose concentration which can totally repress the expression of <span class="italic">RFP</span>.</div>
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                                 According to our expectation, the more arabinose added, the more inhibitor will be expressed. The downstream RFP will be repressed. That’s what we are going to prove.
+
                             <div class="block-paragraph">
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                            </div>
+
                                 We assumed that, more arabinose added, more inhibitor will be expressed and the downstream in-promoter will be repressed. That’s what we are going to prove.
−
                            <div>
+
−
                                But when we added the arabinose, the RFP intensity increased, and it contradicted with expected results. We thought the terminator cannot completely isolate the two devices.
+
−
                                <b style="color: #BD670A;">So we change the promoter direction and add another B0015 to optimize the circuits.</b>
+
 
                             </div>
 
                             </div>
 +
                            <div class="block-paragraph">
 +
                                But when arabinose was added, RFP intensity increased and it contradicted with the expected results. Maybe the terminator can’t completely isolate the two devices. Thought of it this way,
 +
                                <b style="color: #BD670A;">we change the promoter direction and add another B0015 to optimize the circuits.</b>
  
−
                            <div>
 
−
                                <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
 
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                                    alt="fig5" style="width: 60%;" class="center-block">
 
 
                             </div>
 
                             </div>
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                             <div>
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                             <div class="block-paragraph">
−
                                 We set an arabinose concentration gradient: 0, 0.0002％, 0.0005％, 0.0010％, 0.0015％, 0.0020％, 0.0030％, 0.0040％, 0.0050％. Under these concentrations, we measured the OD600 and RFP intensity.
+
                                 The circuits are shown in the Fig.7.
 +
                                <br>So we change the promoter direction and add another B0015 to optimize the circuits.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
−
                                 <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
+
                                 <img src="https://static.igem.org/mediawiki/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
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                                     alt="fig6" style="width: 60%;" class="center-block">
+
                                     alt="fig7" style="width: 100%;" class="center-block">
 
                             </div>
 
                             </div>
−
                             <div>
+
                      <div class="pic_info"><b>Fig.7</b> Optimized circuits after changing the direction of promoter P<sub>BAD</sub> adding another terminator B0015.</div>
−
                                 The improvement of device construction that we added a terminator and changed the promoter direction did work and thus we observed the decrease of RFP intensity when we increase the arabinose concentration. It indicated that the change of arabinose concentration led to the change of inhibitor concentration, and the inhibitor can influence the expression of downstream gene. Here we use RFP gene to replace killer gene.
+
                             <div class="block-paragraph">
 +
                                 After the pre-experiment, we chose a series of appropriate arabinose concentrations, and they are listed in Table.3. The negative control is the strain containing the empty vector <span class="italic">pSB1C3</span> and the positive control is the strain containing the circuits mentioned above with no arabinose added.
 
                             </div>
 
                             </div>
 +
                          <div class="pic_info"><b>Table.3 </b> The concentrations of arabinose added in the experiment. </div>
 
                             <div>
 
                             <div>
−
                                 <img src="https://static.igem.org/mediawiki/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
+
                                 <img src="https://static.igem.org/mediawiki/2016/d/d9/T--BIT-China--Project--Proof--table3.png"
−
                                     alt="fig7" style="width: 60%;" class="center-block">
+
                                     alt="table3" style="width: 70%;" class="center-block">
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                            <div class="block-paragraph">
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                                The improvement of device construction was that we added a terminator and changed the promoter direction. In this way, we could observe the decrease of RFP intensity when the arabinose concentration increases. It indicates that the change of arabinose concentration will affect inhibitor’s concentration, and the inhibitor can influence the expression of downstream gene. We chose the <span class="italic">cI</span>-P<sub>r</sub> circuit to do this experiment and got the diagram describing the relationship between the time and RFP intensity under different concentration of arabinose.
 
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                                 When the arabinose concentration reached to 0.0030％-0.0040％, the RFP can hardly express. The turning point proved that the inhibitor concentration at this point can almost completely repress the killer gene. Also, we proved that
+
                                 <img src="https://static.igem.org/mediawiki/2016/3/32/T--BIT-China--Project--Proof--fig8.png"
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                                 <b style="color: #BD670A;">inhibitor concentration can adjust the expression of killer gene.</b>
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                                    alt="fig8" style="width: 90%;" class="center-block">
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                            </div>
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                            <div class="pic_info"><b>Fig.8</b> RFP intensity measured under different concentration of arabinose.</div>
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                            <div class="block-paragraph">
 +
                                From this diagram we can see, when the arabinose’s concentration reaches to 0.0030％-0.0040％, the RFP can hardly express. The result proved that the inhibitor can almost completely repress the killer gene at the turning point. Also,
 +
                                we could say
 +
                                 <b style="color: #BD670A;">inhibitor concentration can regulate the expression of the killer gene.</b>
 
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                                 Above all, we proved that plasmid numbers will influence the concentration of inhibitor proteins, and the inhibitor concentration will influence the expression of killer gene which is indicated by RFP measurement results. This way, the change of plasmid numbers will influence the expression of killer gene, which means we can sense the plasmid numbers and accordingly decide whether or not to turn on the switch of killer gene.
+
                                 Above all, we proved that plasmid numbers will influence the concentration of inhibitor proteins, and the inhibitor concentration will regulate the expression of killer gene which is indicated by RFP measurement results.
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                                After connecting with killer gene, the plasmids losing on different levels will influence the expression of killer gene, which means we can sense the plasmid numbers and accordingly decide whether or not to turn on the switch of killer gene. From all these, we can achieve the goal of controlling the number of plasmids as we need.
 
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Latest revision as of 16:43, 9 November 2016

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title
To prove our concept that:
(1) We can make the bacteria sense the plasmid numbers.
(2) The in-promoter will respond differently to different signal which can reflect the plasmids losing on different levels. This way, our system can control the plasmids numbers above a threshold.
We designed two parts. One of them is to prove that plasmid numbers will influence the inhibitor concentration. The other one is to prove the inhibitor concentration can regulate the expression of the killer gene by affecting its in-promoter.
Plasmid numbers will influence the inhibitor concentration
Because of the difficulty of controlling the number of plasmids, we can only choose some typical copy numbers of plasmids in our system.
The copy numbers are shown in the table below:
Table.1 The copy numbers of different plasmids.
table1
We also constructed the gene circuits containing constitutive promoters with different strengths to express the inhibitor. By this we regulate the threshold of plasmid numbers to meet different needs.
fig1
Fig.1 The device constructed to express the inhibitor with a constitutive promoter.
There are four promoters with different strengths and two kinds of RBS we have chosen:
Table.2 The strengths and efficiencies of different promoters and RBS.
table1
Meanwhile, the RFP used to replace the inhibitor protein can directly represent the concentration of the inhibitor in the cell. We separately constructed these circuits on different vectors which have different copy numbers.
At first, we use the modeling to explain the relationship of the concentration of the inhibitor and plasmids number like the curve below under a certain condition
曲线图暂时没有1
In the wet experiment, after the same amount of time, we will measure the RFP intensity to get the data which can describe the relationship between the inhibitor concentration and the plasmid copy numbers.
曲线图暂时没有2
The strength of J23106 is stronger J23116, we can clearly know the inhibitor protein have a positive correlation with plasmids number.
This way, we verified that plasmid numbers will influence the inhibitor concentration on the plasmid vector.
The inhibitor concentration can regulate the expression of the killer gene
In order to know the relationship between the inhibitor and in-promoter, we use the arabinose induced promoter PBAD to express the inhibitor. So we can add arabinose with different concentrations to induce the promoter and create an environment with different concentrations of intracellular inhibitor. Killer gene is replaced by RFP under control of in-promoter.
We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
fig4
Fig.4 Arabinose induced expression cassette of three kinds of inhibitors.
Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
fig5
Fig.5 In-promoter controlled expression cassette of RFP. RFP is used to replace killer gene.
We assembled these corresponding circuits together for the final testing.
fig6
Fig.6 Assembly of inhibitor and "in-promoter". They can be used to test the minimum arabinose concentration which can totally repress the expression of RFP.
We assumed that, more arabinose added, more inhibitor will be expressed and the downstream in-promoter will be repressed. That’s what we are going to prove.
But when arabinose was added, RFP intensity increased and it contradicted with the expected results. Maybe the terminator can’t completely isolate the two devices. Thought of it this way, we change the promoter direction and add another B0015 to optimize the circuits.
The circuits are shown in the Fig.7.
So we change the promoter direction and add another B0015 to optimize the circuits.
fig7
Fig.7 Optimized circuits after changing the direction of promoter PBAD adding another terminator B0015.
After the pre-experiment, we chose a series of appropriate arabinose concentrations, and they are listed in Table.3. The negative control is the strain containing the empty vector pSB1C3 and the positive control is the strain containing the circuits mentioned above with no arabinose added.
Table.3 The concentrations of arabinose added in the experiment.
table3
The improvement of device construction was that we added a terminator and changed the promoter direction. In this way, we could observe the decrease of RFP intensity when the arabinose concentration increases. It indicates that the change of arabinose concentration will affect inhibitor’s concentration, and the inhibitor can influence the expression of downstream gene. We chose the cI-Pr circuit to do this experiment and got the diagram describing the relationship between the time and RFP intensity under different concentration of arabinose.
fig8
Fig.8 RFP intensity measured under different concentration of arabinose.
From this diagram we can see, when the arabinose’s concentration reaches to 0.0030％-0.0040％, the RFP can hardly express. The result proved that the inhibitor can almost completely repress the killer gene at the turning point. Also, we could say inhibitor concentration can regulate the expression of the killer gene.
Summary:
Above all, we proved that plasmid numbers will influence the concentration of inhibitor proteins, and the inhibitor concentration will regulate the expression of killer gene which is indicated by RFP measurement results.
After connecting with killer gene, the plasmids losing on different levels will influence the expression of killer gene, which means we can sense the plasmid numbers and accordingly decide whether or not to turn on the switch of killer gene. From all these, we can achieve the goal of controlling the number of plasmids as we need.

P-SLACKiller

· Work or Die
· Monitor Plasmid Numbers
· Kill the Slacker
· Maintain the Better Biosystem

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