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<h3>Killer System</h3>
 
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<p>
 
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Plasmids are self-replicating pieces of DNA harboring functional genes and have been widely used as DNA recombinant tools.
 
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However, the deficiency, plasmid’s stability, has put many scientists into a dilemma. During the experiments, we often find that the recombinant plasmids’ concentration declined with unknown cause.<p>
 
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<p>Although researchers has designed many widely used methods, such as using auxotrophic bacteria and resistance screening, adding regents during the experiment is too convenient. We do have other method called selfish plasmids which use toxin gene and antitoxin gene at the same time. Once the cell becomes plasmid-free, those toxin protein which is more difficult-to-decompose longer will come into play.
 
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Nevertheless, it still has its shortcoming. We don’t know in what situation a cell can be called as plasmid-free? And in another way we can say, what’s the threshold of plasmids’ concentration? Our team is going to optimize the whole system,to make it more accurate, more controllable and intelligent.</p>
 
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<p>So we decide to set up an alarm clock in bacteria with toxin gene and CRISPR Cas which could remind the bacteria of how many plasmids remain to work.
 
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We do hope that our project will not only make great contribution to animalcule fermentation engineering helping saving a large sum of extra manpower and material resources, but also show  you a new research way of measuring the amount of plasmids.
 
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</p>
 
  
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<h5>References</h5>
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        .nav_left_ul{
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
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<h5>Inspiration</h5>
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<p>See how other teams have described and presented their projects: </p>
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<ul>
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
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            height: 60px;
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
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            line-height: 60px;
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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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/2016/5/56/T--BIT-China--Project--Description--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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                <!--Problem we aim to solve-->
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                <div id="to_solve" class="block-title col-sm-12">Problem we aim to solve</div>
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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>
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                                Genetic engineered bacteria which use plasmid as expression vector are widely
 +
                                employed in many social domains and developed rapidly these years. However,
 +
                                the <b>plasmid segregational instability</b> has been the limitation of scientific
 +
                                research and large-scale industry production for plasmid-based expression system.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                So, what’s the plasmid segregational instability?
 +
                            </div>
 +
                            <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>
 +
 +
                <!--Project overview-->
 +
                <div id="overview" class="block-title col-sm-12">Project overview</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-item">
 +
 +
                        <div class="block-content-item-block">
 +
                            <div>
 +
                                We decide to equip the bacteria with a
 +
                                <b>
 +
                                    plasmid-sensing logically adjustable cell killer (P-SLACKiller)
 +
                                </b>
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                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.
 +
                            </div>
 +
                            <div>
 +
                                <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BIT-China--Project--Description--fig7.png"
 +
                                    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>
 +
 +
 +
                <!--Previous work done by others [reference]-->
 +
                <div id="previous" class="block-title">Previous work done by others</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-item">
 +
 +
                        <div class="block-content-item-block">
 +
                            <div>
 +
                                Previous work realize the plasmid maintenance
 +
                                through making the plasmid encode essential factors for the host.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                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 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 class="block-paragraph">
 +
                                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) <sup>[2]</sup>. 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.
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
 +
                <!--Our special design-->
 +
                <div id="special" class="block-title">What’s the difference</div>
 +
                <div class="block-content">
 +
                    <div class="block-content-item">
 +
                        <div class="block-content-item-block">
 +
                            <div class="block-content-header">
 +
                                <i class="fa fa-check-square" aria-hidden="true"></i>&nbsp;Quantitative control
 +
                            </div>
 +
                            <div>
 +
                                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.
 +
                            </div>
 +
                            <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>
 +
                                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.
 +
                            </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>
 +
                                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.
 +
                            </div>
 +
                            <div class="block-paragraph">
 +
                                As an antibiotics-free project, we can improve the previous approaches by
 +
                                making the process of plasmid-control more environment-friendly.
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
 +
                <div class="references">
 +
                    <div style="color:#923F91">
 +
                        <h3>Reference:</h3>
 +
                    </div>
 +
                    <div>
 +
                        [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.
 +
                    </div>
 +
                    <div>
 +
                        [2] Friehs K. Plasmid copy number and plasmid stability. [J]. Advances
 +
                        in Biochemical Engineering/biotechnology, 2004, 86:47-82.
 +
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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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