Difference between revisions of "Team:Tianjin/Design"

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        <meta name="author" content="773715181 Jiaxiao Han">
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        <meta content="2018iGEM Team:Tianjin Kai System to control biological clock" name="description">
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        <title>Team:Tianjin - 2018.igem.org</title>
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#globalWrapper,#content{background-color:transparent;border:0;border-bottom:0;float:none;margin:0;padding:0;width:100%;list-style-image:none}#top_title{margin:0}#HQ_page th{background-color:transparent;padding:0;color:black;border:0;border-collapse:collapse;vertical-align:text-top;text-transform:uppercase;text-align:center}#HQ_page td{padding:0;border:0;vertical-align:text-top;text-align:center;line-height:35px}#HQ_page .jumbotron p{text-align:center;margin-bottom:15px;font-weight:200}#HQ_page p{text-align:center}body{margin-bottom:-10px}#top_title,#sideMenu{display:none}a:hover{text-decoration:none}.mw-content-ltr ul{padding-top:0;padding-right:0;margin:auto}body{margin-top:0}#wrapper{padding-left:0}#page-wrapper{width:100%;padding:0}.intro{background-size:cover;width:100%;position:relative}.huge{font-size:50px;line-height:normal}
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<h1>Design</h1>
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2018.igem.org/Team:Tianjin/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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                    PROJECT
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                <ul class="dropdown-menu" style="left: -14px;">
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Description">BACKGROUND</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Design">DESIGN</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Experiments">EXPERIMENTS</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Notebook">NOTEBOOK</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Model">MODEL</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Measurement">MEASUREMENT</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Demonstrate">DEMONSTRATE</a></li>
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                    PARTS
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Parts">PARTS OVERVIEW</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Basic_Part">BASIC PARTS</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Composite_Part">COMPOSITE PARTS</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Part_Collection">PART COLLECTION</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Improve">IMPROVED PART</a></li>
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                <a href="https://2018.igem.org/Team:Tianjin/Safety">
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                    SAFETY             
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Human_Practices">INTEGRATED</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Collaborations">COLLABORATION</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Public_Engagement">PUBLIC ENGAGEMENT</a></li>
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                    <li><a href="https://2018.igem.org/Team:Tianjin/Attributions">ATTRIBUTIONS</a></li>
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                <a href="https://2018.igem.org/Team:Tianjin/Judging">
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                    FOR JUDGES             
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                    <p>PROJECT DESIGN</p>
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              Circadian clocks, also know as circadian oscillators,&nbsp;are ubiquitous timing systems that induce rhythms of biological&nbsp;activities in synchrony with night and day.&nbsp;Circadian oscillators are post-translationally regulated and affect gene expression in autotrophic circadian cyanobacteria.&nbsp;Most work on the cyanobacterial&nbsp;circadian&nbsp;clock has been performed in <em>Synechococcus elongatus</em>&nbsp;PCC&nbsp;7942 (<em>S. elongatus</em>). In <em>S. elongatus</em>&nbsp;7942, timing is generated by a&nbsp;post-translational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output&nbsp;signaling proteins, SasA, RpaA and CikA, which transduce this rhythm to control gene expression&nbsp;and chromosome topology <sup><a href="#re1">1</a></sup>.
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                  The goal of our project is to reconstruct the KaiABC circadian clock system from prokaryotic cyanobacteria (<em>S.</em><em>&nbsp;</em><em>elongatus</em>&nbsp;7942) in noncircadian eukaryotic <em>Saccharomyces cerevisiae</em>&nbsp;(<em>S</em><em>.</em><em>&nbsp;cerevisiae</em>). And we are aim to achieve the controllability of this circadian clock system in Saccharomyces cerevisiae&nbsp;through some practical methods, such as changing the molecular concentration ratio of the core protein&nbsp;KaiA, KaiB and KaiC, which can help us better understand and futher explore the KaiABC system. In addition to the reconstruction of KaiABC, we tried to regulate the metabolic activities&nbsp;of <em>S</em><em>.</em><em>&nbsp;cerevisiae</em>&nbsp;using this prokaryotic circadian&nbsp;clock system. What’s more, Our project got a lot of meaningful inspiration from our modeling work. According to the reaction mechanism, a series of rate equations can be obtained. And our modeling work has successfully influenced and corrected our understanding of the KaiABC rhythm system. Click <a href="https://2018.igem.org/Team:Tianjin/Model">here</a> for more imformation about Modeling.
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                  Some previous research findings have pointed to a possible global regulatory mechanism of the clock through the systematic alteration of chromosome topology<sup><a href="#re2">2</a></sup>. Although the underlying process are not revealed yet, researchers have identified that <em>S. elongatus</em>&nbsp;PCC 7942 genes exhibit circadian fluctuations and that these changes are highly correlated with rhythmic changes in the superhelical density of the chromosome<sup><a href="#re3">3</a></sup>.&nbsp;Inspired by these studies, we expect that the <em>S. cerevisiae</em>&nbsp;chromasome&nbsp;topology will periodically oscillate with the KaiABC circadian clock system.
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                  In addition to understanding and exploring basic biological science through construction&nbsp;and building, we also explored applications of this circadian clock system in <em>S</em><em>.</em><em>&nbsp;cerevisiae</em>. The novel application we envisioned was that <em>S</em><em>.</em><em>&nbsp;cerevisiae</em>&nbsp;can produce different products alternately under the periodic regulation&nbsp;of the KaiABC circadian clock.
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<li>Explanation of the engineering principles your team used in your design</li>
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<li>Discussion of the design iterations your team went through</li>
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                                1. Reconstruction of KaiABC circadian clock with different strategies
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                                      In order to enable the reconstitution system of the selected six proteins, KaiA KaiB KaiC RpaA CiKA and SasA, to successfully work in yeast cells, we used three different strategies to assemble the gene expression cassettes of these six proteins onto two plasmids. At the same time, in order to better activate the reporter gene circuits to express the oscillating signal, we explored the interaction of three different couples of proteins in the system with the control of the circadian clock system.
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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                                2. Design of gene circuits and output of oscillating signals
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                                      In order to affect cellular physiology, a posttranslational circadian clock needs to be connected to transcriptional output. To do so, we built a synthetic oscillator circuit and introduced a yeast two-hybrid system. <br>
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                                      Firstly, we utilized the synthetic oscillator circuit to control the expression of a selected reporter gene. In order to select a suitable reporter gene, we tested some of the parameters of several commonly used reporter genes by pre-experiment and scored these reporter genes by modeling.
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                                3. Regulation of introduced prokaryotic clock systems in eukaryotic cells
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                                      Based on the information reviewed so far, changing the concentration of the protein or using protein mutants can change the oscillating signal. We control the concentration of expressed protein by using different promoters. And The ability of the promoter to initiate expression was determined by experimental assays.
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                                4. Prokaryotic clock system regulates eukaryotic yeast by periodically affecting the topology of eukaryotic chromatin
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                                    <p>
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                                      S. elongatus genes exhibit circadian fluctuations and that these changes are highly correlated with rhythmic changes in the superhelical density of the chromosome . Although the underlying process are not revealed yet, some previous research findings have pointed to a possible global regulatory mechanism of the clock through the systematic alteration of chromosome topology.<br>
 +
                                      Based on this background knowledge, we designed experiments to couple the regulation of the KaiABC rhythm oscillation system with the S. cerevisiae chromatin topology. It is possible to achieve the regulation of the transcription and expression of global endogenous genes by KaiABC rhythm system in yeast. We choosed chromatin remodeling complex for research.<sup><a href="#re6">6</a></sup>
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                              5. Novel applications
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                                      Our Tik Tok S. cerevisiae, which has been introduced into the KaiABC biological clock system, can be used as a cell factory that alternates between day and night to produce different products, which contributes to a good work schedule.<br>
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                    <h1>References</h1>
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                    <p class="reftext" id="re1">
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                        <a>[1]Tseng, R. et al. Structural basis of the day-night transition in a bacterial circadian clock. Science 355, 1174-1180, doi:10.1126/science.aag2516 (2017).</a>
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                        <a>[2]Shultzaberger, R. K., Boyd, J. S., Diamond, S., Greenspan, R. J. & Golden, S. S. Giving Time Purpose: The Synechococcus elongatus Clock in a Broader Network Context. Annu Rev Genet 49, 485-505, doi:10.1146/annurev-genet-111212-133227 (2015).</a>
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                    </p>
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                    <p class="reftext" id="re3">
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                        <a>[3]Woelfle, M. A., Xu, Y., Qin, X. & Johnson, C. H. Circadian rhythms of superhelical status of DNA in cyanobacteria. Proc Natl Acad Sci U S A 104, 18819-18824, doi:10.1073/pnas.0706069104 (2007).</a>
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                    </p>
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                    <p class="reftext" id="re4">
 +
                        <a>[4]Kageyama, H. et al. Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro. Mol Cell 23, 161-171, doi:10.1016/j.molcel.2006.05.039 (2006).</a>
 +
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                    </p>
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                    <p class="reftext" id="re5">
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                        <a>[5]Terauchi, K. et al. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. P Natl Acad Sci USA 104, 16377-16381, doi:10.1073/pnas.0706292104 (2007).</a>
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                    <p class="reftext" id="re6">
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                        <a>[6]Narlikar, G. J., Fan, H. Y. & Kingston, R. E. Cooperation between complexes that regulate chromatin structure and transcription. Cell 108, 475-487, doi:Doi 10.1016/S0092-8674(02)00654-2 (2002).</a>
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                    <p class="reftext" id="re7">
 +
                        <a>[7]Rust, M. J., Golden, S. S. & O'Shea, E. K. Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator. Science 331, 220-223, doi:10.1126/science.1197243 (2011).</a>
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Latest revision as of 13:00, 6 December 2018

<!DOCTYPE html> Team:Tianjin - 2018.igem.org

PROJECT DESIGN

Circadian clocks, also know as circadian oscillators, are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. Circadian oscillators are post-translationally regulated and affect gene expression in autotrophic circadian cyanobacteria. Most work on the cyanobacterial circadian clock has been performed in Synechococcus elongatus PCC 7942 (S. elongatus). In S. elongatus 7942, timing is generated by a post-translational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA, RpaA and CikA, which transduce this rhythm to control gene expression and chromosome topology 1.

The goal of our project is to reconstruct the KaiABC circadian clock system from prokaryotic cyanobacteria (S. elongatus 7942) in noncircadian eukaryotic Saccharomyces cerevisiae (S. cerevisiae). And we are aim to achieve the controllability of this circadian clock system in Saccharomyces cerevisiae through some practical methods, such as changing the molecular concentration ratio of the core protein KaiA, KaiB and KaiC, which can help us better understand and futher explore the KaiABC system. In addition to the reconstruction of KaiABC, we tried to regulate the metabolic activities of S. cerevisiae using this prokaryotic circadian clock system. What’s more, Our project got a lot of meaningful inspiration from our modeling work. According to the reaction mechanism, a series of rate equations can be obtained. And our modeling work has successfully influenced and corrected our understanding of the KaiABC rhythm system. Click here for more imformation about Modeling.

Some previous research findings have pointed to a possible global regulatory mechanism of the clock through the systematic alteration of chromosome topology2. Although the underlying process are not revealed yet, researchers have identified that S. elongatus PCC 7942 genes exhibit circadian fluctuations and that these changes are highly correlated with rhythmic changes in the superhelical density of the chromosome3. Inspired by these studies, we expect that the S. cerevisiae chromasome topology will periodically oscillate with the KaiABC circadian clock system.

In addition to understanding and exploring basic biological science through construction and building, we also explored applications of this circadian clock system in S. cerevisiae. The novel application we envisioned was that S. cerevisiae can produce different products alternately under the periodic regulation of the KaiABC circadian clock.

1. Reconstruction of KaiABC circadian clock with different strategies
2. Design of gene circuits and output of oscillating signals
3. Regulation of introduced prokaryotic clock systems in eukaryotic cells
4. Prokaryotic clock system regulates eukaryotic yeast by periodically affecting the topology of eukaryotic chromatin
5. Novel applications

References

[1]Tseng, R. et al. Structural basis of the day-night transition in a bacterial circadian clock. Science 355, 1174-1180, doi:10.1126/science.aag2516 (2017).

[2]Shultzaberger, R. K., Boyd, J. S., Diamond, S., Greenspan, R. J. & Golden, S. S. Giving Time Purpose: The Synechococcus elongatus Clock in a Broader Network Context. Annu Rev Genet 49, 485-505, doi:10.1146/annurev-genet-111212-133227 (2015).

[3]Woelfle, M. A., Xu, Y., Qin, X. & Johnson, C. H. Circadian rhythms of superhelical status of DNA in cyanobacteria. Proc Natl Acad Sci U S A 104, 18819-18824, doi:10.1073/pnas.0706069104 (2007).

[4]Kageyama, H. et al. Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro. Mol Cell 23, 161-171, doi:10.1016/j.molcel.2006.05.039 (2006).

[5]Terauchi, K. et al. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. P Natl Acad Sci USA 104, 16377-16381, doi:10.1073/pnas.0706292104 (2007).

[6]Narlikar, G. J., Fan, H. Y. & Kingston, R. E. Cooperation between complexes that regulate chromatin structure and transcription. Cell 108, 475-487, doi:Doi 10.1016/S0092-8674(02)00654-2 (2002).

[7]Rust, M. J., Golden, S. S. & O'Shea, E. K. Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator. Science 331, 220-223, doi:10.1126/science.1197243 (2011).