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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                         To prove our concept that
+
                         To prove our concept that:
−
                        <a href="https://2016.igem.org/Team:BIT-China/Results" style="color:blue">See our final results here!</a>
+
 
                     </div>
 
                     </div>
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                     <div>
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                     <div class="block-paragraph">
 
                         (1) We can make the bacteria sense the plasmid numbers.
 
                         (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.
+
                         <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>
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                     <div>
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                     <div class="block-paragraph">
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                         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".
+
                         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 style="color:#923F91;margin-top: 20px" id="influence">
 
                             <div style="color:#923F91;margin-top: 20px" id="influence">
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                                 <i class="fa fa-magic" aria-hidden="true"></i>
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−
                                <span class="block-content-header">
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                                <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>
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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.
 
                                 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:
 
                                 <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>
 
                                 <img src="https://static.igem.org/mediawiki/2016/9/9f/T--BIT-China--Project--Proof--table1.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/9/9f/T--BIT-China--Project--Proof--table1.png"
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                                     alt="table1" class="center-block" style="width: 400px;">
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                                     alt="table1" class="center-block" style="width: 300px;">
 
                             </div>
 
                             </div>
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                             <div>
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                             <div class="block-paragraph">
 
                                 We also constructed the gene circuits containing constitutive promoters with
 
                                 We also constructed the gene circuits containing constitutive promoters with
 
                                 different strengths to express the inhibitor. By this we regulate the threshold
 
                                 different strengths to express the inhibitor. By this we regulate the threshold
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                             <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"
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                                     alt="fig1" class="center-block" style="height: 100px;">
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                                     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>
 +
                             <div class="block-paragraph">
 
                                 There are four promoters with different strengths and two kinds of RBS we have chosen:
 
                                 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>
 
                                 <img src="https://static.igem.org/mediawiki/2016/4/44/T--BIT-China--Project--Proof--table2.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/4/44/T--BIT-China--Project--Proof--table2.png"
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                                     alt="table1" class="center-block" style="height: 150px;">
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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">
 
                                 Meanwhile, the RFP used to replace the inhibitor protein can directly represent the
 
                                 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.
 
                                 concentration of the inhibitor in the cell. We separately constructed these circuits on different vectors which have different copy numbers.
−
                                <br>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>
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                             <div>
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                             <div class="block-paragraph">
−
                                <img src="" alt="曲线图暂时没有1" style="width:60%" class="center-block">
+
                                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>
 +
                                <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.
 
                                 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>
−
                                 <img src="" alt="曲线图暂时没有2" style="width:60%" class="center-block">
+
                                 <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>
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                             <div>
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                             <div class="block-paragraph">
−
                                 This way, we verified that plasmid numbers will influence the inhibitor concentration under on the plasmid vector.
+
                                 This way, we verified that plasmid numbers will influence the inhibitor concentration on the plasmid vector.
 
                             </div>
 
                             </div>
  
  
 
                             <div style="color:#923F91;margin-top: 20px" id="adjust">
 
                             <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">
 +
                                <i class="fa fa-magic" aria-hidden="true"></i>
 
                                     The inhibitor concentration can regulate the expression of the killer gene
 
                                     The inhibitor concentration can regulate the expression of the killer gene
 
                                 </span>
 
                                 </span>
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
−
                                 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".
+
                                 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.
−
                                <br>We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
+
 
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
 
                                 <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
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                                     alt="fig4" style="height: 250px;" 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>
−
                                 Meanwhile, we designed three corresponding "in-promoter" circuits in Fig.5.
+
                             <div class="block-paragraph">
 +
                                 Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
 
                                 <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
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                                     alt="fig5" style="height: 300px;" class="center-block">
+
                                     alt="fig5" style="width: 50%;" class="center-block">
 
                             </div>
 
                             </div>
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                             <div>
+
                             <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>
 
                                 <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
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                                     alt="fig6" style="height: 300px;" class="center-block">
+
                                     alt="fig6" style="width: 100%;" class="center-block">
 
                             </div>
 
                             </div>
−
                             <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>
−
                                 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 class="block-paragraph">
 +
                                 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>
 
                             </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,
 
                                 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>
 
                                 <b style="color: #BD670A;">we change the promoter direction and add another B0015 to optimize the circuits.</b>
 +
 +
                            </div>
 +
                            <div class="block-paragraph">
 
                                 The circuits are shown in the Fig.7.
 
                                 The circuits are shown in the Fig.7.
−
                                 So we change the promoter direction and add another B0015 to optimize the circuits.
+
                                 <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/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
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                                     alt="fig7" style="height: 250px;" 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>
 +
                             <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.
 
                                 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/d/d9/T--BIT-China--Project--Proof--table3.png"
 
                                 <img src="https://static.igem.org/mediawiki/2016/d/d9/T--BIT-China--Project--Proof--table3.png"
−
                                     alt="table3" style="height: 350px;" class="center-block">
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                                     alt="table3" style="width: 70%;" class="center-block">
 
                             </div>
 
                             </div>
−
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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 cI-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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                                 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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                             <div class="pic_info"><b>Fig.8</b> RFP intensity measured under different concentration of arabinose.</div>
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                                 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,
 
                                 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
 
                                 we could say
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                                 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.
 
                                 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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                                <br>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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                                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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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.

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