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

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{{BIT-China}}
 
{{BIT-China}}
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<div class="column full_size judges-will-not-evaluate">
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#mw-content-text{
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
 
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<p>
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        .block-title{
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iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.  
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        .problem-title{
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<h4> What should we do for our proof of concept? </h4>
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You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
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<div class="col-sm-12 clearfix" style="padding: 0;padding-top:30px;
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background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)">
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      <div class="content-right" style="float: left;width:100%;padding: 10px;">
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        <img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--content_decoration.png"
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            <div class="overview-content clearfix">
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                <div class="content-title col-sm-12">
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                    <img src="https://static.igem.org/mediawiki/2016/2/20/T--BIT-China--Project--Proof--title.png"
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                        alt="title" class="col-sm-8 col-sm-offset-2">
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                    <!--<br>-->
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                </div>
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                <!--问题描述-->
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                <div class="problem-txt block-content col-sm-12" style="margin-top: 10px">
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                    <div>
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                        To prove our concept that:
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                    </div>
 +
                    <div class="block-paragraph">
 +
                        (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>
 +
 +
                <!--Problem we aim to solve-->
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                <div class="block-content">
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                    <div class="block-content-item">
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                        <div class="block-content-item-block">
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                            <div style="color:#923F91;margin-top: 20px" id="influence">
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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
 +
                                </span>
 +
                            </div>
 +
                            <div>
 +
                                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>
 +
                                <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 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>
 +
                                <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%;">
 +
                            </div>
 +
                          <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:
 +
                            </div>
 +
                          <div class="pic_info"><b>Table.2</b> The strengths and efficiencies of different promoters and RBS.</div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/4/44/T--BIT-China--Project--Proof--table2.png"
 +
                                    alt="table1" class="center-block" style="width: 90%;">
 +
                            </div>
 +
 +
                            <div class="block-paragraph">
 +
                                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>
 +
                                <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>
 +
                                <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 style="color:#923F91;margin-top: 20px" id="adjust">
 +
                                <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
 +
                                </span>
 +
                            </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.
 +
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/3/33/T--BIT-China--Project--Proof--fig4.png"
 +
                                    alt="fig4" style="width: 60%;" class="center-block">
 +
                            </div>
 +
                          <div class="pic_info"><b>Fig.4</b> Arabinose induced expression cassette of three kinds of inhibitors.</div>
 +
                            <div class="block-paragraph">
 +
                                Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/0/0a/T--BIT-China--Project--Proof--fig5.png"
 +
                                    alt="fig5" style="width: 50%;" class="center-block">
 +
                            </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.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Proof--fig6.png"
 +
                                    alt="fig6" style="width: 100%;" class="center-block">
 +
                            </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>
 +
                            <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 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>
 +
                            <div class="block-paragraph">
 +
                                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>
 +
                                <img src="https://static.igem.org/mediawiki/2016/0/05/T--BIT-China--Project--Proof--fig7.png"
 +
                                    alt="fig7" style="width: 100%;" class="center-block">
 +
                            </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.
 +
                            </div>
 +
                          <div class="pic_info"><b>Table.3 </b> The concentrations of arabinose added in the experiment. </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/d/d9/T--BIT-China--Project--Proof--table3.png"
 +
                                    alt="table3" style="width: 70%;" class="center-block">
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                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.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/3/32/T--BIT-China--Project--Proof--fig8.png"
 +
                                    alt="fig8" style="width: 90%;" class="center-block">
 +
                            </div>
 +
                            <div class="pic_info"><b>Fig.8</b> RFP intensity measured under different concentration of arabinose.</div>
 +
                            <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>
 +
                            </div>
 +
 +
                            <div style="color:#923F91;margin-top: 20px" id="summary">
 +
                                <i class="fa fa-magic" aria-hidden="true"></i>
 +
                                <span class="block-content-header">
 +
                                    Summary:
 +
                                </span>
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                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.
 +
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                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.
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
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            </div>
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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.

P-SLACKiller

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

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