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

Revision as of 09:13, 16 October 2016

Proof of concept

To prove our concept that See our final results here!

(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 number of the copy numbers are shown in the table below:

We also constructed the gene circuits containing constitutive promoters with different strengths to express the inhibitor. By this we regulate the threshold of plasmids number to meet different needs.

There are four promoters with different strengths and two kinds of RBS we have choose:

Meanwhile, the RFP used to replace the inhibitor protein can directly represent the concentration of the inhibitor in the cell. We separately constructed there 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

And then after passing a period 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.

This way, we verified that plasmid numbers will influence the inhibitor concentration under on the plasmid vector.

The inhibitor concentration can regulate the expression of the killer gene

In order to know the relationship of 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 providing an environment with different concentrations of intracellular inhibitor. Kill gene is replaced by RFP to stand the in-promoter’s status.
We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.

Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.

We assembled these corresponding circuits together for the final testing.

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 contradicting 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.9. So we change the promoter direction and add another B0015 to optimize the circuits.

After the pre-experiment, we chose a series of appropriate arabinose concentrations, and they are listed in table.1. The negative control is the strain containing the empty vector pSB1C3 and the positive control is the strain containing the (序号) with no arabinose added.

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 of the relationship of the time and RFP intensity 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 kill 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.

Proof

Influence
Adjust
Summary

@@ Line 109: / Line 109: @@
 <div class="col-sm-12 clearfix" style="padding: 0;padding-top:30px;
 background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)">
-    <div class="content-right" style="float: left;width:100%;padding: 10px;">
+        <div class="content-right" style="float: left;width:100%;padding: 10px;">
          <div style="margin-left:220px;background: white;margin-top: 0.5em;margin-bottom:0.5em;padding: 1px;border-radius: 10px">
              <div class="overview-content" style="border:1px dashed #F08B1F;
@@ Line 124: / Line 124: @@
                      <div class="problem-title">Proof of concept</div>
                      <div>
-                         <span>
+                         To prove our concept that
-                            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.
+                        <a href="https://2016.igem.org/Team:BIT-China/Results">See our final results here!</a>
-                            <a href="https://2016.igem.org/Team:BIT-China/Results">See our final results here!</a>
+                    </div>
-                        </span>
+                    <div>
+                        (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>
+                        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>
@@ Line 135: / Line 140: @@
                      <div class="block-content-item">
                          <div class="block-content-item-block">
-                             <div style="color:#923F91;" id="influence">
+                             <div style="color:#923F91;margin-top: 20px" id="influence">
                                  <i class="fa fa-magic" aria-hidden="true"></i>
                                  <span class="block-content-header">
@@ Line 142: / Line 148: @@
                              </div>
                              <div>
-                                 We use plasmids with different copies to adjust the plasmid numbers. To better simulate the real situation for application, we employed the constitutive promoter.
+                                 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 number of the copy numbers are shown in the table below:
                              </div>
                              <div>
-                                 We built devices containing constitutive promoters with different strengths.
+                                 <img src="https://static.igem.org/mediawiki/2016/9/9f/T--BIT-China--Project--Proof--table1.png"
+                                     alt="table1" class="center-block" style="width: 400px;">
                              </div>
+                            <div>
+                                We also constructed the gene circuits containing constitutive promoters with different strengths to express the inhibitor. By this we regulate the threshold of plasmids number to meet different needs.
+                            </div>
                              <div>
                                  <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="height: 100px;">
                              </div>
                              <div>
-                                 Meanwhile, we <b style="color: #BD6C13;">use RFP to replace the inhibitor gene</b> to indicate the inhibitor concentration.
+                                 There are four promoters with different strengths and two kinds of RBS we have choose:
-                                We have chosen plasmids with different copies and separately ligated these circuits on the vectors.
                              </div>
                              <div>
-                                 <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"
-                                      alt="fig2" style="width: 40%;" class="center-block">
+                                      alt="table1" class="center-block" style="height: 150px;">
                              </div>
                              <div>
-                                 Through testing, we got data describing the relationship between the inhibitor concentration and plasmid copies.
+                                 Meanwhile, the RFP used to replace the inhibitor protein can directly represent the concentration of the inhibitor in the cell. We separately constructed there 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>
-                                 <img src="" alt="曲线图暂时没有">
+                                 <img src="" alt="曲线图暂时没有1" style="width:60%" class="center-block">
                              </div>
                              <div>
-                                 This way, we verified that plasmid numbers will influence the inhibitor concentration.
+                                And then after passing a period 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>
+                                <img src="" alt="曲线图暂时没有2" style="width:60%" class="center-block">
+                            </div>
+                            <div>
+                                 This way, we verified that plasmid numbers will influence the inhibitor concentration under on the plasmid vector.
                              </div>
-                             <div style="color:#923F91;" id="adjust">
+                             <div style="color:#923F91;margin-top: 20px" id="adjust">
                                  <i class="fa fa-magic" aria-hidden="true"></i>
                                  <span class="block-content-header">
-                                     Inhibitor concentration will adjust the expression of killer gene
+                                     The inhibitor concentration can regulate the expression of the killer gene
                                  </span>
                              </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 of 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 providing an environment with different concentrations of intracellular inhibitor. Kill gene is replaced by RFP to stand the in-promoter’s status.
+                                <br>We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
                              </div>
                              <div>
-                                 We built three devices containing different kinds of inhibitors.
+                                 <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
+                                     alt="fig4" style="height: 250px;" class="center-block">
                              </div>
                              <div>
-                                 <img src="https://static.igem.org/mediawiki/2016/3/3d/T--BIT-China--Project--Proof--fig22.png"
+                                 Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
-                                     alt="fig2" style="width: 60%;" class="center-block">
                              </div>
                              <div>
-                                Meanwhile, we built three corresponding in-promoter circuits and used RFP to replace the killer gene.
+                                 <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
-                            </div>
+                                      alt="fig5" style="height: 300px;" class="center-block">
-                            <div>
-                                 <img src="https://static.igem.org/mediawiki/2016/0/0f/T--BIT-China--Project--Proof--fig3.png"
-                                      alt="fig3" style="width: 60%;" class="center-block">
                              </div>
                              <div>
@@ Line 199: / Line 214: @@
                              </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/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
-                                      alt="fig4" style="width: 60%;" class="center-block">
+                                      alt="fig6" style="height: 300px;" class="center-block">
                              </div>
                              <div>
-                                 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.
+                                 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>
-                                 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.
+                                 But when arabinose was added, RFP intensity increased contradicting with the expected results.
-                                 <b style="color: #BD670A;">So we change the promoter direction and add another B0015 to optimize the circuits.</b>
+                                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>
+                                The circuits are shown in the Fig.9.
+                                So we change the promoter direction and add another B0015 to optimize the circuits.
                              </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/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
-                                      alt="fig5" style="width: 60%;" class="center-block">
+                                      alt="fig7" style="height: 250px;" class="center-block">
                              </div>
                              <div>
-                                 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.
+                                 After the pre-experiment, we chose a series of appropriate arabinose concentrations, and they are listed in table.1. The negative control is the strain containing the empty vector pSB1C3 and the positive control is the strain containing the (序号) with no arabinose added.
                              </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/d/d9/T--BIT-China--Project--Proof--table3.png"
-                                      alt="fig6" style="width: 60%;" class="center-block">
+                                      alt="table3" style="height: 350px;" class="center-block">
                              </div>
                              <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.
+                                 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 of the relationship of the time and RFP intensity under different concentration of arabinose.
                              </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/2016/3/32/T--BIT-China--Project--Proof--fig8.png"
-                                      alt="fig7" style="width: 60%;" class="center-block">
+                                      alt="fig8" style="height: 420px;" class="center-block">
                              </div>
                              <div>
-                                 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
+                                 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,
-                                 <b style="color: #BD670A;">inhibitor concentration can adjust the expression of killer gene.</b>
+                                we could say
+                                 <b style="color: #BD670A;">inhibitor concentration can regulate the expression of the killer gene.</b>
                              </div>
+                             <div style="color:#923F91;margin-top: 20px" id="summary">
-                             <div style="color:#923F91;" id="summary">
                                  <i class="fa fa-magic" aria-hidden="true"></i>
                                  <span class="block-content-header">
@@ Line 241: / Line 258: @@
                              </div>
                              <div>
-                                 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.
+                                <br>After connecting with kill 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.
                              </div>
                          </div>