Difference between revisions of "Team:NKU CHINA"

 
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</div>
 
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        <div class="fullscreenvideo" >
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        <video id="videohome" autoplay="autoplay" muted="muted" loop="loop">
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          <source src="https://static.igem.org/mediawiki/2018/d/db/T--NKU_CHINA--home_bg.mp4" type="video/mp4">
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        </video>
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        </div>
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<main>
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    <img src="https://static.igem.org/mediawiki/2018/9/98/T--NKU_CHINA--PopQC_larger.gif" id="PopQC_largr" style="position:absolute;left: 28%;top: -14%;z-index: 10;width: 600px;height: auto;">
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    <h1>Description</h1>
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            <p><br><br></p>
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    <P>0. Abstract</P>
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          </div>
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    <p>Synthetic biology has been used to produce various compounds, yet its’ yield still await improvements to become economical. It has been revealed that in a single colony, different cells have considerably variations in protein and metabolite concentrations due to some nongenetic differences, suggesting the existence of both high- and low-performance variants in all cultures. Our project is aimed to design an in vivo population quality control system(PopQc) to continuously select high-performing, nongenetic variants and in that improving the yield of target products. In the presence of PopQc, we could accurately select cells producing glutamate above a settled level by changing the concentration of tetracycline, which could give a notable improvement to the yield of glutamate.</p>
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        </div>
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    <P>1. Motivation</P>
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      </div>
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    <P>By means of gene manipulation, microorganisms, especially bacteria, have been widely used as bio-factories to synthesize various kinds of significant products ranging from simple fuels (for example, ethanol, butanol and fatty acid derivatives) to intricate natural products (artemisinin, strictosidine, erythromycin, and so on). However, one great weakness of biosynthesis compared to petroleum-based chemical synthesis is that biosynthesis often tends to be uneconomical. Its’ relatively low productivity and yield have suppressed it future development. So, it’s necessary to create new approaches to enhance biosynthetic performance.</P>
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      <div class="container">
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<P>Nongenetic variation emerging in microbial cultures is known to be attributed to naturally inherent factors including uneven cell division, epigenetic modifications and variations in gene copy numbers, random gene expression and variant mRNA stabilities and protein activities. The overall effect of these factors, no matter in plasmid-based or in chromosome-based gene expression, could generate a considerably range of variation in protein and metabolite concentrations, suggesting a potential approach to improve biosynthetic yields. So based on that, we developed in vivo PopQc system.</P>
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        <div class="row">
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<P>2. PopQc</P>
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          <div class="col-xs-12">
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<P>What is PopQc? PopQc is the logogram of population quality control system. This system selects high-performing cells by the product-mediated regulation of transcription of antibiotic resistance gene. In the presence of antibiotic, while high-performing cells could produce antibiotic-resistant proteins properly and maintain alive, low-performing cells due to the low concentration of target product in vivo, are suppressed to express antibiotic-resistant proteins and ends in death.
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            <h2 style="color: white; font-size: 60px; font-family: myTitle3;text-align: center;margin-top:30px;margin-bottom:20px;padding-top:0;padding-bottom:0;" id="dinwei">Short Video Display</h2>
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In our assays, we choose Bacillus amyloliquefaciens LL3 as the host to be engineered, and our aim is to select cells in which the concentration of intracellular glutamate is high.
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          </div>
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</P>
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          <div class="col-xs-12 col-xs-push-1">
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<P>3. Introduction of Bacillus amyloliquefaciens LL3</P>
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              <video preload="preload" controls="controls" width="800" height="auto" id="nku_video" poster="https://static.igem.org/mediawiki/2018/b/b2/T--NKU_CHINA--video_image.jpg">
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<P>Bacillus amyloliquefaciens LL3, which is a gram-positive bacterium, was originally separated from traditional fermented foods by prof. Song’s lab. Here are the main characteristics making it suitable to be the host.
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              <source src="https://static.igem.org/mediawiki/2018/b/b6/T--NKU_CHINA--igem_NKU.mp4" type="video/mp4">
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First, it has a clear genetic background, because our lab has already finished its’ genome sequencing in 2011. Second, its’ synthesis of poly-γ-glutamate (γ-PGA) is independent of exogenous glutamate.  
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            </video>
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</P>
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          </div>
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<P>4. Our design</P>
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          <div class="col-xs-12" style="margin-top: 10px;">
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<P>To achieve the selection of high-performing cells, our project is aiming to apply PopQc in Bacillus amyloliquefaciens LL3.
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              <p style="text-align: center;"><a href="https://youtu.be/Gmr6zBYlpK0" style="color: white;font-size: 25px;">Cilck to watch our video on <span style="color: yellow;">YouTube</span></a></p>
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Here is the basic mechanism of PopQc we designed. Transcription factor GltC, positively regulated by α-ketoglutarate and negatively regulated by glutamate, could enhance the promoter PgltAB, which is followed by lacI, the gene of protein LacI. LacI could bind promoter PlacO，then suppress the expression of the following tetA, gene of tetracycline discharge pump. So, assuming the outer tetracycline concentration is at an even level, when the glutamate concentration in the cell achieves a certain level, the cell will have the ability to pump out tetracycline to stay alive. On the contrast, in low-perform cells, the suppression of tetA expression would lead these cells to death.
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          </div>
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After designing this system, we also need to quantify it. So, we designed some further assays to examine.
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            <p style="color: white;font-size: 23px;line-height: 30px;" style="float: left;text-align: justify;">Population Quality Control (PopQC) system, is a new approach designed for biosynthesis yield enhancement, based on non-genetic cell-to-cell variation, which includes unequal cell division, different gene copy numbers, epigenetic modifications, random gene expression, volatile RNA stability, protein activity, etc.</p>
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            <p style="color: white;font-size: 23px;line-height: 30px;text-align: justify;">Because of those differences, different cells in a single colony will have considerable variations in protein and metabolite concentrations. Therefore, in cell cultures there will be both high- and low-producers, and the intrinsic low-producers might cause suboptimal ensemble biosynthesis. The elimination of low-producers can realize the efficient utilization of substrates and high yield of target products. Thus, it has been proved to be an efficient way for biosynthesis being more suitable for large-scale industrial production. In our project, PopQC was designed as a plasmid-based gene circuit in <i>Bacillus amyloliquefaciens</i> LL3, which continuously selects high-performing cells in order to further improve the yield of target metabolite— glutamate, and then the secondary metabolites— &#947;-PGA.</p>
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Population Quality Control (PopQC) system, is a new approach designed for biosynthesis yield enhancement, based on non-genetic cell-to-cell variation, which includes unequal cell division, different gene copy numbers, epigenetic modifications, random gene expression, volatile RNA stability, protein activity, etc.

Because of those differences, different cells in a single colony will have considerable variations in protein and metabolite concentrations. Therefore, in cell cultures there will be both high- and low-producers, and the intrinsic low-producers might cause suboptimal ensemble biosynthesis. The elimination of low-producers can realize the efficient utilization of substrates and high yield of target products. Thus, it has been proved to be an efficient way for biosynthesis being more suitable for large-scale industrial production. In our project, PopQC was designed as a plasmid-based gene circuit in Bacillus amyloliquefaciens LL3, which continuously selects high-performing cells in order to further improve the yield of target metabolite— glutamate, and then the secondary metabolites— γ-PGA.

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Nankai University
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Tianjin, P.R.China 300071

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