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

 
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     position: relative;
 
     position: relative;
 
}
 
}
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/*页内左侧导航*/
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             font-weight: bold;
 
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 +
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.block-paragraph{
 +
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 +
<script>
  
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</head>
 
</head>
 
<body>
 
<body>
  
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<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)">
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<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;">
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         <div style="margin-left:220px;background: white;margin-top: 0.5em;margin-bottom:0.5em;padding: 1px;border-radius: 10px">
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−
            <div class="overview-content" style="border:1px dashed #F08B1F;
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         <img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--content_decoration.png"
−
             border-radius: 10px;margin: 0.5em;padding: 10px;">
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            alt="content_decoration" style="position:absolute;right: 0px;height: 150px;;top: 10px;">
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 +
        <div class="block-border-outer" id="content-right-border">
 +
             <div class="overview-content clearfix">
  
 
                 <div class="content-title col-sm-12">
 
                 <div class="content-title col-sm-12">
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                     Description
+
                     <img src="https://static.igem.org/mediawiki/2016/5/56/T--BIT-China--Project--Description--title.png"
−
                    <!--<img src="https://static.igem.org/mediawiki/2016/3/30/T&#45;&#45;BIT-China&#45;&#45;Project&#45;&#45;Design&#45;&#45;title.png"-->
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                         alt="title" class="col-sm-8 col-sm-offset-2">
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                         <!--alt="title" class="col-sm-8 col-sm-offset-2">-->
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                     <br>
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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">
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−
                    <div class="problem-title">Proof of concept</div>
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−
                    <div>
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−
                        <img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"
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−
                            alt="ul_logo1" width="18" height="18">
+
−
                        <span>
+
−
                            We have successfully proved our concept of plasmid-sensing logically
+
−
                            adjustable cell killer
+
−
                            (P-SLACKiller) at every step of our model and wet experiment.
+
−
                            <a href="##">See our final results here!</a>
+
−
                        </span>
+
−
                    </div>
+
 
                 </div>
 
                 </div>
  
 
                 <!--Problem we aim to solve-->
 
                 <!--Problem we aim to solve-->
−
                 <div id="to_solve" class="block-title">Problem we aim to solve</div>
+
                 <div id="to_solve" class="block-title col-sm-12">Problem we aim to solve</div>
 
                 <div class="block-content">
 
                 <div class="block-content">
 
                     <div class="block-content-item">
 
                     <div class="block-content-item">
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                         <div class="block-content-item-block">
 
                         <div class="block-content-item-block">
 
                             <div>
 
                             <div>
−
                                 For plasmid-based expression system, the
+
                                 Genetic engineered bacteria which use plasmid as expression vector are widely
−
                                 <b>plasmid segregational instability</b>
+
                                employed in many social domains and developed rapidly these years. However,
−
                                has been the limit step in both scientific research
+
                                 the <b>plasmid segregational instability</b> has been the limitation of scientific
−
                                 and large-scale industry production. Generation of plasmid-free
+
                                 research and large-scale industry production for plasmid-based expression system.
−
                                cells will sharply decrease the efficiency and
+
−
                                profit due to <b>the slacker’s</b> increasing proliferation ability
+
−
                                while producing no target substances.
+
 
                             </div>
 
                             </div>
−
                             <div>
+
                             <div class="block-paragraph">
−
                                 Enhancing the plasmid stability is our main goal.
+
                                 So, what’s the plasmid segregational instability?
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                                 To control the intracellular plasmid numbers,
+
                            </div>
−
                                 the first step is to <b>sense</b> the plasmid numbers.
+
                            <div class="block-paragraph">
 +
                                It’s known that uneven cell division arises frequently which is bound to
 +
                                 produce two daughter cells with different plasmid numbers. The cell with
 +
                                less plasmids will produce cells with much less plasmids.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                In our project, those cells with not enough plasmid number are called
 +
                                 <b>"Slacker"</b>, on the contrary, <b>"Worker"</b> is the name for cells with enough plasmid.
 +
                            </div>
 +
                            <div class="col-sm-12" style="margin: 10px auto;">
 +
                                <img src="https://static.igem.org/mediawiki/2016/1/1a/T--BIT-China--Project--Description--fig1.png"
 +
                                    alt="fig1" class="col-sm-4">
 +
                                <img src="https://static.igem.org/mediawiki/2016/1/14/T--BIT-China--img--home_overview1.png"
 +
                                    alt="fig1" class="col-sm-4">
 +
                                <img src="https://static.igem.org/mediawiki/2016/6/60/T--BIT-China--img--home_overview2.png"
 +
                                    alt="fig1" class="col-sm-4">
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                The existence of slackers will sharply decrease the efficiency and profit due to the slacker’s
 +
                                increasing proliferation ability while producing no target substances.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                There is no efficient way to prevent the birth of the slackers. Can we make
 +
                                the bacteria themselves monitor the plasmid losing situation and eliminate these slackers?
 +
                                In this way, we can enhance the plasmid stability,
 +
                                stabilize the microbial population structure and finally improve the production efficiency.
 +
                            </div>
 +
 
 +
                            <div class="col-sm-12" style="margin: 10px auto;">
 +
                                <img src="https://static.igem.org/mediawiki/2016/5/51/T--BIT-China--Project--Description--fig4.png"
 +
                                    alt="fig4" class="col-sm-4">
 +
                                <img src="https://static.igem.org/mediawiki/2016/8/8d/T--BIT-China--img--home_overview4.png"
 +
                                    alt="fig5" class="col-sm-4">
 +
                                <img src="https://static.igem.org/mediawiki/2016/4/47/T--BIT-China--img--home_overview5.png"
 +
                                    alt="fig6" class="col-sm-4">
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
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                 <!--Project overview-->
 
                 <!--Project overview-->
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                 <div id="overview" class="block-title">Project overview</div>
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                 <div id="overview" class="block-title col-sm-12">Project overview</div>
 
                 <div class="block-content">
 
                 <div class="block-content">
 
                     <div class="block-content-item">
 
                     <div class="block-content-item">
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                         <div class="block-content-item-block">
 
                         <div class="block-content-item-block">
 
                             <div>
 
                             <div>
−
                                 Equip the bacteria with a
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                                 We decide to equip the bacteria with a
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                                 <b>plasmid-sensing logically adjustable cell killer (P-SLACKiller)</b>,
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                                 <b>
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                                we select the inhibitor
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                                    plasmid-sensing logically adjustable cell killer (P-SLACKiller)
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                                protein as a signal which can indicate the intracellular plasmid numbers.
+
                                </b>
 
                             </div>
 
                             </div>
−
                             <div>
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                             <div class="block-paragraph">
−
                                 There is a basic rule: when the plasmid numbers are above the threshold,
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                                 In our project, the inhibitor protein’s concentration is used as a signal to indicate the
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                                we regard it as a normally-working bacterium and the P-SLACKiller won’t
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                                 intracellular plasmid numbers. The sketch map of our basic circuit is shown in Fig.1.
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                                 start; however, when the plasmid numbers are
+
−
                                below the threshold, we judge it as a slacker and the P-SLACKiller
+
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                                will kill these slackers.
+
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
−
                                 <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig1.png"
+
                                 <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Description--fig7.png"
−
                                     alt="fig1" class="center-block" style="width: 60%;">
+
                                     alt="fig1" class="center-block" style="width:80%">
 +
                                <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.1</b> The basic circuit of P-SLACKiller.</div>
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                <br>We choose a constitutive promoter to express inhibitor gene, and an "in-promoter" which
 +
                                is repressed by inhibitor to express killer gene. Under normal circumstances, for
 +
                                plasmid concentration being high enough, our killer is repressed by inhibitor whose
 +
                                concentration is connected with plasmid numbers. However, considering the effect of
 +
                                plasmid segregational instability, the plasmid numbers will decrease, so as the
 +
                                intracellular inhibitor protein. The inadequate inhibitor cannot completely repress the
 +
                                expression of downstream killer gene, and all those slackers will be killed.
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
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                 <!--Previous work done by others [reference]-->
 
                 <!--Previous work done by others [reference]-->
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                 <div id="previous" class="block-title">Previous work done by others [reference]</div>
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                 <div id="previous" class="block-title">Previous work done by others</div>
 
                 <div class="block-content">
 
                 <div class="block-content">
 
                     <div class="block-content-item">
 
                     <div class="block-content-item">
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                                 through making the plasmid encode essential factors for the host.
 
                                 through making the plasmid encode essential factors for the host.
 
                             </div>
 
                             </div>
−
                             <div>
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                             <div class="block-paragraph">
−
                                 Resistance screening and auxotrophic bacteria are dependent on the selecting process [1].
+
                                 Resistance screening and auxotrophic bacteria are depended on the selecting process <sup>[1]</sup>.
−
                                 The plasmid-free bacteria cannot survive under specific selections, such as <b>antibiotics</b>.
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                                 The plasmid-free bacteria cannot survive under specific selections, such as antibiotics.
 +
                                However, it’s not applicable for industrial fermentation. What’s worse, the unlimited
 +
                                use of antibiotics has raised worldwide concern due to its potential risk which
 +
                                may cause antibiotic resistance,
 +
                                degrade the capacity of the immune system and finally develop to a big social problem.
 
                             </div>
 
                             </div>
−
                             <div>
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                             <div class="block-paragraph">
−
                                 According to the natural mechanisms for plasmid maintenance,
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                                 According to the natural mechanisms for plasmid maintenance, many new strategies are
−
                                many new strategies are developed, so called
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                                developed, so called plasmid addiction system (PAS) or post-segregational killing
−
                                <b>plasmid addiction system
+
                                system (PSK) <sup>[2]</sup>. However, these mechanisms are still under exploration and the
−
                                    (PAS) or post-segregational
+
                                capacity of plasmid maintenance is not ideal since most of them can only ensure
−
                                    killing system (PSK)</b>
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                                one plasmid remains, can’t truly realize the quantitative control as we hope.
−
                                [2]. However, these mechanism are still under exploration.
+
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
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                 <!--Our special design-->
 
                 <!--Our special design-->
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                 <div id="special" class="block-title">Our special design</div>
+
                 <div id="special" class="block-title">What’s the difference</div>
 
                 <div class="block-content">
 
                 <div class="block-content">
 
                     <div class="block-content-item">
 
                     <div class="block-content-item">
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                                <i class="fa fa-check-square" aria-hidden="true"></i>&nbsp;Quantitative control
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                            </div>
 
                             <div>
 
                             <div>
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                                 As we know, the employed antibiotics
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                                 Most existing methods for enhancing biosynthetic performance are stuck
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                                 <b>must be removed</b>
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                                at the level of resistance screening or rely on natural mechanisms
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                                in pharmaceutical or GMP-based
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                                 for plasmid maintenance. These methods have the same problem: there is
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                                 fermentation processes and it’s not an
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                                no clear definition for high- and low-performance variants, or said differently,
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                                 applicable option in industrial fermentation.
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                                those methods can’t define a number or a range as the threshold of plasmids’ concentration.
 +
                            </div>
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                            <div class="block-paragraph">
 +
                                 In our project, we can not only set a threshold to control the number of plasmids, but also
 +
                                 regulate it as we need by using the different biobricks. That is what we say—quantitative control.
 +
                            </div>
 +
 
 +
 
 +
                            <div class="block-content-header">
 +
                                <i class="fa fa-check-square" aria-hidden="true"></i>&nbsp;No human intervention
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 In our project, we aim to use an intracellular signal correlated with
+
                                 There is a step called spawn rejuvenation in the existing large-scale
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                                 the plasmid numbers and realize the selection process without any human interference.
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                                industrial production procedure.
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                                 This design will greatly <b>simplify the spawn rejuvenation</b>.
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                                It’s useful to maintain the microbial population structure, but costs plenty of time and money.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                Our design has great potential to simplify the process of spawn rejuvenation
 +
                                as well as increase the profit for companies. We use an intracellular signal
 +
                                 correlated with the plasmid numbers, and realize the selection process without
 +
                                any human interference. Through keeping the plasmid numbers above a threshold,
 +
                                 we can realize the population quality control and improve the production efficiency.
 +
                            </div>
 +
 
 +
                            <div class="block-content-header">
 +
                                <i class="fa fa-check-square" aria-hidden="true"></i>&nbsp;No antibiotics
 
                             </div>
 
                             </div>
 
                             <div>
 
                             <div>
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                                 <b>With no antibiotics added</b>, we also want to improve the previous
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                                 As we know, the employed antibiotics must be removed in pharmaceutical or GMP-based fermentation
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                                 approaches by realizing the <b>quantification of plasmid copy numbers</b>.
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                                processes and it’s not an applicable option in industrial fermentation.
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                                <a href="##">
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                            </div>
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                                    See our design.</a>
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                            <div class="block-paragraph">
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                                As an antibiotics-free project, we can improve the previous approaches by
 +
                                 making the process of plasmid-control more environment-friendly.
 
                             </div>
 
                             </div>
 
                         </div>
 
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                         <h3>Reference:</h3>
 
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                         [1] Kroll J, Klinter S, Schneider C, et al. Plasmid addiction systems: perspectives and
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                         [1] Kroll J, Klinter S, Schneider C, et al. Plasmid addiction systems:
 +
                        <br>perspectives and
 
                         applications in biotechnology. [J]. Microbial Biotechnology, 2010, 3(6):634-57.
 
                         applications in biotechnology. [J]. Microbial Biotechnology, 2010, 3(6):634-57.
 
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Latest revision as of 16:14, 9 November 2016

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Problem we aim to solve
Genetic engineered bacteria which use plasmid as expression vector are widely employed in many social domains and developed rapidly these years. However, the plasmid segregational instability has been the limitation of scientific research and large-scale industry production for plasmid-based expression system.
So, what’s the plasmid segregational instability?
It’s known that uneven cell division arises frequently which is bound to produce two daughter cells with different plasmid numbers. The cell with less plasmids will produce cells with much less plasmids.
In our project, those cells with not enough plasmid number are called "Slacker", on the contrary, "Worker" is the name for cells with enough plasmid.
fig1 fig1 fig1
The existence of slackers will sharply decrease the efficiency and profit due to the slacker’s increasing proliferation ability while producing no target substances.
There is no efficient way to prevent the birth of the slackers. Can we make the bacteria themselves monitor the plasmid losing situation and eliminate these slackers? In this way, we can enhance the plasmid stability, stabilize the microbial population structure and finally improve the production efficiency.
fig4 fig5 fig6
Project overview
We decide to equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller)
In our project, the inhibitor protein’s concentration is used as a signal to indicate the intracellular plasmid numbers. The sketch map of our basic circuit is shown in Fig.1.
fig1
Fig.1 The basic circuit of P-SLACKiller.

We choose a constitutive promoter to express inhibitor gene, and an "in-promoter" which is repressed by inhibitor to express killer gene. Under normal circumstances, for plasmid concentration being high enough, our killer is repressed by inhibitor whose concentration is connected with plasmid numbers. However, considering the effect of plasmid segregational instability, the plasmid numbers will decrease, so as the intracellular inhibitor protein. The inadequate inhibitor cannot completely repress the expression of downstream killer gene, and all those slackers will be killed.
Previous work realize the plasmid maintenance through making the plasmid encode essential factors for the host.
Resistance screening and auxotrophic bacteria are depended on the selecting process [1]. The plasmid-free bacteria cannot survive under specific selections, such as antibiotics. However, it’s not applicable for industrial fermentation. What’s worse, the unlimited use of antibiotics has raised worldwide concern due to its potential risk which may cause antibiotic resistance, degrade the capacity of the immune system and finally develop to a big social problem.
According to the natural mechanisms for plasmid maintenance, many new strategies are developed, so called plasmid addiction system (PAS) or post-segregational killing system (PSK) [2]. However, these mechanisms are still under exploration and the capacity of plasmid maintenance is not ideal since most of them can only ensure one plasmid remains, can’t truly realize the quantitative control as we hope.
What’s the difference
 Quantitative control
Most existing methods for enhancing biosynthetic performance are stuck at the level of resistance screening or rely on natural mechanisms for plasmid maintenance. These methods have the same problem: there is no clear definition for high- and low-performance variants, or said differently, those methods can’t define a number or a range as the threshold of plasmids’ concentration.
In our project, we can not only set a threshold to control the number of plasmids, but also regulate it as we need by using the different biobricks. That is what we say—quantitative control.
 No human intervention
There is a step called spawn rejuvenation in the existing large-scale industrial production procedure. It’s useful to maintain the microbial population structure, but costs plenty of time and money.
Our design has great potential to simplify the process of spawn rejuvenation as well as increase the profit for companies. We use an intracellular signal correlated with the plasmid numbers, and realize the selection process without any human interference. Through keeping the plasmid numbers above a threshold, we can realize the population quality control and improve the production efficiency.
 No antibiotics
As we know, the employed antibiotics must be removed in pharmaceutical or GMP-based fermentation processes and it’s not an applicable option in industrial fermentation.
As an antibiotics-free project, we can improve the previous approaches by making the process of plasmid-control more environment-friendly.

Reference:

[1] Kroll J, Klinter S, Schneider C, et al. Plasmid addiction systems:
perspectives and applications in biotechnology. [J]. Microbial Biotechnology, 2010, 3(6):634-57.
[2] Friehs K. Plasmid copy number and plasmid stability. [J]. Advances in Biochemical Engineering/biotechnology, 2004, 86:47-82.

P-SLACKiller

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

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Email: iGEM_BIT_China@163.com
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