View source for Team:Peking/Project

<html lang="en">

<head>

    <style>


        /***************************************************** DEFAULT WIKI SETTINGS  ****************************************************/

        /* Clear the default wiki settings */

        #home_logo, #sideMenu {
            display: none;
        }

        #sideMenu, #top_title, .patrollink {
            display: none;
        }

        #content {
            width: 100%;
            padding: 0px;
            margin-top: 0px;
            margin-left: 0px;
        }

        body {
            background-color: #FAFAFA;
        !important;
        }

        #bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 {
            margin-bottom: 0px;
        }

        ol, ul {
            list-style: none
        }

        ol li:before {
            content: counter(ol) ".";
            counter-increment: ol;
            color: #02A9F7;
            text-align: right;
            display: inline-block;
            min-width: 1em;
            margin-right: 0.5em
        }

        ol ol {
            margin-left: 1.25714em
        }

        ol li {
            text-indent: -1.95em;
            line-height: 2em;
        }

        .nonumberitem ul li {
            text-indent: -0.65em;
            line-height: 2em;
        }

        .nonumberitem ul li:before {
            content: "•";
            color: #02A9F7;
            display: inline-block;
            margin-right: 0.3em;
        !important;
        }

        ul ul {
            margin-left: 1.25714em
        }


    </style>

    <link rel="stylesheet" href="https://fonts.googleapis.com/icon?family=Material+Icons">
    <script type="text/javascript" src="https://2017.igem.org/Template:Peking/mdl/materialminjs?
action=raw&ctype=text/javascript"></script>
    <link href="https://2017.igem.org/Template:Peking/mdl/component?action=raw&ctype=text/css" rel="stylesheet">
    <script type="text/javascript"
            src="https://2017.igem.org/Template:Peking/mdl/componentjs?action=raw&ctype=text/javascript"></script>

    <link rel="stylesheet" type="text/css"
          href="https://2017.igem.org/Template:Peking/mdl/MDLnew?action=raw&ctype=text/css">

    <link rel="stylesheet" type="text/css"
          href="https://2017.igem.org/Template:Peking/mdl/main?action=raw&ctype=text/css">
    <!-- Fonts -->
    <link rel="stylesheet" type="text/css"
          href="https://2017.igem.org/Template:Peking/mdl/fonts?action=raw&ctype=text/css">
    <link rel="stylesheet" type="text/css"
          href="https://2017.igem.org/Template:Peking/mdl/icon?action=raw&ctype=text/css">


</head>


<body>


<!-- Always shows a header, even in smaller screens. -->
<div class="mdl-layout mdl-js-layout mdl-layout--fixed-header">
    <header class="mdl-layout__header" style="position:fixed; margin-top: -7px; !important;">
        <div class="mdl-layout__header-row">
            <!-- Title -->
            <a href="https://2017.igem.org/Team:Peking"><img
                    src="https://static.igem.org/mediawiki/2017/c/cf/Peking_TeamLogoClassical.png" height="35"></a>

            <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking"
               style="color: #000000; font-size: x-large"><strong>Peking iGEM </strong> 2017</a>
            <!-- Add spacer, to align navigation to the right -->
            <div class="mdl-layout-spacer"></div>
            <!-- Navigation. We hide it in small screens. -->
            <nav class="mdl-navigation mdl-layout--large-screen-only">
                <style>
                    a:link {
                        text-decoration: none;
                    }

                    a:visited {
                        text-decoration: none;
                    }

                    a:active {
                        text-decoration: none;
                    }

                    a:hover {
                    }
                </style>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking">Home</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Project#Introduction"
                   style="color: #000; font-weight: 500 ">Project</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Model#Overview">Modelling</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Software">Software</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Hardware">Hardware</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Lab">Lab</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/HP">Practices</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Parts#Overview">Parts</a>
                <a class="mdl-navigation__link" href="https://2017.igem.org/Team:Peking/Team">Team</a>
            </nav>
        </div>
    </header>

</div>

<main class="docs-layout-content mdl-layout__content mdl-color-text--grey-600"
      style="width: 100%; padding-bottom: 0px;">
    <div class="content mdl-grid mdl-grid--no-spacing" id="content">
        <div class="mdl-components mdl-js-components mdl-cell mdl-cell--12-col"
             style="padding-top: 60px; background-color: #FAFAFA">
            <aside class="mdl-components__nav docs-text-styling mdl-shadow--4dp">

                <a href="https://2017.igem.org/Team:Peking/Project#Introduction"
                   class="mdl-components__link mdl-component Introduction">
                    <div class="mdl-components__link-image"
                         style="background-image: url(https://static.igem.org/mediawiki/2017/5/55/Peking_Background_icon.png)">
                    </div>
                    <span class="mdl-components__link-text" style="font-size: medium">Introduction</span>
                </a>


                <a href="https://2017.igem.org/Team:Peking/Project#Flip-flop"
                   class="mdl-components__link mdl-component FF">
                    <div class="mdl-components__link-image"
                         style="background-image: url(https://static.igem.org/mediawiki/2017/1/1a/Peking_FF_icon.png)">
                    </div>
                    <span class="mdl-components__link-text" style="font-size: medium">Flip-flop</span>
                </a>

                <a href="https://2017.igem.org/Team:Peking/Project#Controller"
                   class="mdl-components__link mdl-component CU">
                    <div class="mdl-components__link-image"
                         style="background-image: url(https://static.igem.org/mediawiki/2017/1/1d/Peking_Controller_icon.png)">
                    </div>
                    <span class="mdl-components__link-text" style="font-size: medium">Controller</span>
                </a>

                <a href="https://2017.igem.org/Team:Peking/Project#Clock"
                   class="mdl-components__link mdl-component Clock">
                    <div class="mdl-components__link-image"
                         style="background-image: url(https://static.igem.org/mediawiki/2017/5/56/Peking_Clock_icon.svg)">
                    </div>
                    <span class="mdl-components__link-text" style="font-size: medium">Clock</span>
                </a>

                <a href="https://2017.igem.org/Team:Peking/Project#Demonstrate"
                   class="mdl-components__link mdl-component Demonstrate">
                    <div class="mdl-components__link-image"
                         style="background-image: url(https://static.igem.org/mediawiki/2017/8/83/Peking_Demonstrate_icon.png)">
                    </div>
                    <span class="mdl-components__link-text" style="font-size: medium">Demonstrate</span>
                </a>


            </aside>
            <style>
                .demo-card-wide.mdl-card {

                    margin-top: 50px;
                    margin-left: 40px;
                    margin-right: 40px;
                    margin-bottom: 80px;

                    padding-top: 40px;
                    padding-left: 50px;
                    padding-right: 30px;
                    padding-bottom: 40px;

                    width: auto;

                }

                .demo-card-wide > .mdl-card__title {
                    color: #fff;
                    height: 176px;

                }

                .demo-card-wide > .mdl-card__menu {
                    color: #000;
                }

                .demo-card-wide > .mdl-card__supporting-text {
                    /*text-indent: 3em;*/
                    color: rgba(0, 0, 0, 0.85);
                    font-size: 1rem;
                    line-height: 32px;
                    font-family: "Roboto", "Helvetica", "Arial", sans-serif;
                    text-align: justify;
                !important;

                }
            </style>
            <section id="index-section" class="mdl-components-index mdl-components__page is-active">

            </section>
            <section id="Introduction" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h1>Background</h1>
                        <br>

                        <br>

                        Cells are responsive to a myriad signals under most conditions[1]. Upon receiving this
                        cornucopia of signals, i.e. inputs from the external and internal environment, cells must
                        adjust their own internal mechanisms in response in order to survive. This adjustment depends
                        not only on processing a combination of current environmental signal inputs[1], but also on
                        determining the cell’s current state, which is a result of a series of past inputs. In
                        dig-ital circuit theory, this operating mode is known as sequential logic[3].
                        <strong>Sequential logic is a type of logic in which output is a function of the present value
                            of inputs and the system’s internal state.</strong>
                        <br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/f/f7/Peking_description_fig1.png"
                                                 height="300px"/>
                        </div>
                        <br>
                        The field of synthetic biology aims to design biological systems to perform tasks to better
                        understand analogous natural systems and to develop direct applications for research and
                        medicine. Nowadays, a wide va-riety of tasks can be performed by synthetically engineered
                        genetic circuits, mostly constructed using combinational logic. The output of combinational
                        logic (also referred to as time-independent logic) is a function of the present input only[4].
                        In contrast to sequential logic, it has no internal state as an influence of the history of
                        inputs. Without a memory of state, it is difficult to perform functions in a specific order,
                        which has limited the widespread implementation of such systems[5].
                        <br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/3/3a/Peking_description_fig2.png"
                                                 height="300px"/>
                        </div>
                        <br>

                        The ability of sequential logic circuits to store modest amounts of information within living
                        systems and to act upon them would enable new ap-proaches to the study and control of biological
                        processes[6]. Indeed, the attention paid to this field has progressed far beyond scarce. For
                        example, genetic toggle switches have proved to be effective in building synthetic memory
                        modules[7]. In 2010, a bistable push-on push-off switch was constructed as a memory module by
                        Lou et al[8][9]. Five years later, Pakpoom Subsoontorn et al. designed a toggle flip-flop switch
                        whose state can be changed by the same signal[10]. Moreover, in 2012, Jerome Bonnet
                        demon-strated
                        a rewriteable recombinase-addressable data (RAD) module[11]. Re-cently, a state machine with two
                        inputs and five states depending on the order of the input was realized in E. coli by Roquet,
                        N. et al[12].
                        <br><br>

                        These studies indicate that more functions could be accomplished with a memory module embedded
                        within the circuit[13]. Once the memory system is able to scale up to more states, a cell can be
                        designed to do work that is more complex. In other words, we can reach a new dimensionality in
                        designing synthetic life–time.
                        <br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/bf/Peking_description_fig3.png"
                                                 height="300px"/>
                        </div>

                        <br><br>

                        <h1>Introduction</h1>

                        This year, the Peking iGEM team is attempting to develop a frame of biological sequential
                        circuits that are programmable. The envisioned circuit is capable of both storing states in
                        DNA and automatically running a series of instructions in a specific order.
                        <strong>More specifically</strong>,
                        the sequential logic (or a real-state machine) that consists of a clock (trigger signal), flip
                        flop (remembering device) and control unit (functional part) in bacteria. The clock is an
                        oscillator with a repeated signal cycle that is utilized like a metronome to trigger actions
                        of sequential logic circuits. Flip-flop is a memory device that can remember a state. Paired
                        with a clock signal, it can realize state transition. The control unit is a functional part
                        which can convert a signal from flip-flop into complex functions. With such a design,
                        historical events are recorded and influence current cell behavior.
                        <br><br>

                        This work tries to point the way toward building large computational systems from modular
                        biological parts—basic sequential computing devices in living cells, and ultimately, programming
                        complex biological functions. Computers have thus become "alive". A unicellular organism itself
                        cannot pack much computational power, but considered as a modular building block, its potential
                        is impressive. 
                        <br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/0/0a/Peking_description_fig4.png"
                                                 height="300px"/>
                        </div>
                        <br>
                        <br>

                        <h1>References</h1>
                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Hansen, J., & Benenson, Y. (2016). Synthetic biology of cell signaling. Natural
                                Computing, 15(1), 5-13.
                            </li>
                            <li>Densmore, D., & Anderson, J. C. (2009, May). Combinational logic design in synthetic
                                biology. In Circuits and Systems, 2009. ISCAS 2009. IEEE International Symposium on (pp.
                                301-304). IEEE.
                            </li>
                            <li>Cavanagh, Joseph (2006). Sequential Logic: Analysis and Synthesis. CRC Press. pp. ix.
                                ISBN 0849375649.
                            </li>
                            <li>C.J. Savant, Jr.; Martin Roden; Gordon Carpenter. "Electronic Design: Circuits and
                                Systems". 1991. ISBN 0-8053-0285-9 p. 682
                            </li>
                            <li>Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off
                                switch. Mol. Syst. Biol. 6, 350 (2010).
                            </li>
                            <li>Zhang, H., Lin, M., Shi, H., Ji, W., Huang, L., Zhang, X., ... & Yang, Z. (2014).
                                Programming a Pavlovian-like conditioning circuit in Escherichia coli. Nature
                                communications, 5, 3102.
                            </li>
                            <li>Subsoontorn P, Endy D (2012) Design and analysis of genetically encoded counters.
                                Procedia Comput Sci 11:43–54. doi:10.1016/j.procs.2012.09.006CrossRefGoogle Scholar
                            </li>
                            <li>J. Bonnet, P. Subsoontorn, D. Endy, Rewritable digital data storage in live cells via
                                engineered control of recombination directionality. Proc. Natl. Acad. Sci. U.S.A. 109,
                                8884–8889 (2012). 10.1073/pnas.1202344109pmid:22615351 doi:10.1073/pnas.1202344109
                            </li>
                            <li>Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S. & Lu, T.K. Synthetic
                                recombinase-based state machines in living cells. Science 353, aad8559 (2016).
                            </li>
                            <li>Burrill, D. R., & Silver, P. A. (2010). Making cellular memories. Cell, 140(1), 13-18.
                            </li>

                        </ol>


                        <br><br><br><br><a
                            class="mdl-button mdl-button--accent mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                            href="#top" style="color: white;">
                        Back to top
                    </a>


                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->
                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>
                </div>

            </section>
            <section id="Design" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">

                    <div class="mdl-card__supporting-text">

                        <a href="https://2017.igem.org/Team:Peking/Parts#Overview" target="blank" style="color: #226CE4">
                            <h1>Basic Parts</h1></a>

                        Well-characterized and functionally independent recombinase and RDF devices.<br><br>

                        <a href="https://2017.igem.org/Team:Peking/Protocols" target="blank" style="color: #226CE4"><h1>
                            Tools and approaches</h1></a>

                        Standardize the procedure of constructing and characterizing genetic information storage
                        systems.<br><br>

                        <a href="https://2017.igem.org/Team:Peking/Software" target="blank" style="color: #226CE4"><h1>
                            Design</h1></a>

                        Guide construction of framework with sequential function involving the above parts and
                        structures.<br><br>

                        <a href="https://2017.igem.org/Team:Peking/Model#Overview" target="blank" style="color: #226CE4">
                            <h1>Prospect</h1></a>

                        Predict system performance with modeling techniques.<br><br>

                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->
                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">
                    <div class="mdl-card__title"
                         style="background: url('https://static.igem.org/mediawiki/2017/0/08/Peking_design_fig1.jpeg') center / cover;">
                        <h1 class="mdl-card__title-text" style="text-shadow:2px 2px 8px #070707; font-size: xx-large">
                            Flipflop</h1>
                    </div>
                    <div class="mdl-card__supporting-text"
                         style="line-height: 2em;text-align: justify; color: #3A3A3A; padding-left: 50px; padding-top: 50px; padding-bottom:50px">

                        Prove feasibility of bio-flip-flop and optimize its partial function.<br>
                        <ul type=circle style="line-height: 2em;">
                            <li>Select effective recombinases and their corresponding RDFs to characterize</li>
                            <li>Fine-tuning basic parts</li>
                            <li>Construct half of the flip-flop structure and express it in <i>E. coli.</i></li>
                        </ul>
                        <br><br>

                        <a class="mdl-button mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                           href="https://2017.igem.org/Team:Peking/Project#Flip-flop" target="_blank"
                           style="background-color: #2D8EE8; color: white;">
                            Read More
                        </a>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">
                    <div class="mdl-card__title"
                         style="background: url('https://static.igem.org/mediawiki/2017/2/26/Peking_design_fig2.jpeg') center / cover;">
                        <h1 class="mdl-card__title-text" style="text-shadow:2px 2px 8px #070707; font-size: xx-large">
                            Clock</h1>
                    </div>
                    <div class="mdl-card__supporting-text"
                         style="line-height: 2em;text-align: justify; color: #3A3A3A; padding-left: 50px; padding-top: 50px; padding-bottom:50px">

                        Use the repressilator as an internal clock to trigger recombinase expression. Proove the
                        viability of repressilator-triggered bio-flip-flop.<br>
                        <ul type=circle style="line-height: 2em;">
                            <li>Evaluate the repressilator system in our own experimental environment. Show that stable
                                oscillation in fluorescent protein expression can be observed.
                            </li>
                            <li>Construct an oscillator-induced protein expression system and compare its oscillation
                                period with repressilator.
                            </li>
                        </ul>
                        <br><br>

                        <a class="mdl-button mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                           href="https://2017.igem.org/Team:Peking/Project#Clock" target="_blank"
                           style="background-color: #2D8EE8; color: white;">
                            Read More
                        </a>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">
                    <div class="mdl-card__title"
                         style="background: url('https://static.igem.org/mediawiki/2017/c/c4/Peking_design_fig3.jpeg') center / cover;">
                        <h1 class="mdl-card__title-text" style="text-shadow:2px 2px 8px #070707; font-size: xx-large">
                            Control Unit</h1>
                    </div>
                    <div class="mdl-card__supporting-text"
                         style="line-height: 2em;text-align: justify; color: #3A3A3A; padding-left: 50px; padding-top: 50px; padding-bottom:50px">

                        Construct sequences controlling target gene expression, whose pattern is temporally manipulated
                        by recombinases and RDFs.<br>
                        <ul type=circle style="line-height: 2em;">
                            <li>Characterize a group of terminators and measure their strength in both orientations.
                            </li>
                            <li>Select unidirectional terminators that can serve as switches in genetic circuits.</li>
                            <li>Measure the strength of terminators flanked by two sets of attB/P sites, and the
                                inversion efficiency of different recombinases.
                            </li>
                            <li>Construct a seven-segment display which can count from 1 to 3.</li>
                        </ul>
                        <br><br>

                        <a class="mdl-button mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                           href="https://2017.igem.org/Team:Peking/Project#Controller" target="_blank"
                           style="background-color: #2D8EE8; color: white;">
                            Read More
                        </a>
                    </div>

                </div>


            </section>
            <section id="Flip-flop" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h1>Flip-flop</h1>
                        <br>

                        <section class="docs-toc docs-text-styling" style="margin-left: 0px">
                            <nav class="section-content" style="margin-left: 0px">
                                <ul style="margin-left: 0px">

                                    <li><a href="#p1">Background</a></li>

                                    <li><a href="#p2">Description</a></li>

                                    <li><a href="#p3">Key Achievements</a></li>
                                    <li><a href="#p4">Our Approach and Designs</a></li>
                                    <li><a href="#p5">Standard Characterization & Optimization Workflow</a></li>
                                    <li><a href="#p6">Construction of bio-flip-flops</a></li>
                                    <li><a href="#p7">Results</a></li>
                                    <li><a href="#p8">References</a></li>


                                </ul>
                            </nav>
                        </section>


                        <h2 id="p1">Background</h2>
                        The basic building blocks of sequential logic circuits are flip-flops, which are devices that
                        can store information. A flip-flop has two stable states (see more on "<a href="#State"
                                                                                                  style="color: #226CE4"><strong>state</strong></a>")
                        and its state
                        can be changed by signals of arriving pulses. Thus, it and can store one bit of information
                        and alternate its state following a trigger. It is the basic storage element in sequential
                        logic.

                        <a href="#Tflipflop" style="color: #226CE4"><strong>Click here to get a more specific
                            description</strong></a>
                        <br><br>


                        When designing genetic-circuit counterparts of flip-flops, we should consider the following
                        three criteria:<br>
                        <div class="nonumberitem">
                            <ul>
                                <li>The device should have at least two stable states;</li>
                                <li>State changes are triggered by an identical signal;</li>
                                <li>The flip-flop should hold its state and remain unaltered until the next signal
                                    arrives.
                                </li>
                            </ul>
                        </div>

                        In a living cell, DNA is the natural medium for storing cell-state information and encoding
                        functions. Recombinases, especially a subset called serine integrases and excisionases, are
                        enzymes that can flip or excise specific fragments of DNA. They have been proved to be able to
                        stably modify DNA sequences , which is the biological basis of our genetic flip-flop
                        construct.<br><br>
                        Large serine integrases reliably and irreversibly flip or excise unique fragments of DNA . DNA
                        cleavage and re-ligation occur at the central crossover region at a pair of recombinase
                        recognition sites (attB and attP), which allows the sequence to be flipped, excised, or inserted
                        between recognition sites . After recombination, the original attB and attP sequences become
                        reconstructed sequences - attL and attR. The resulting attL and attR sequences cannot be
                        recognized and bound by integrases alone, so the state after integration is stable.<br><br>


                        Another kind of recombinases, excisionases, are able to recognize and bind attL and attR
                        sequences. With the help of excisionases, the state transitions become reversible.<br><br>
                        Recombination Directionality Factors (RDF) are proteins which mediate reverse flipping to
                        recover the sequence flipped by recombinases. By co-expressing the integrase with the
                        corresponding RDF, or expressing the integrase-RDF fusion protein, recombination between the
                        attL and attR sites can be induced. Thus, the flip-flop can be restored to the previous
                        state.<br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/0/0b/Peking_flipflop_fig1.svg"
                                                 height="400px"/>
                        </div>
                        <br>

                        <strong>Figure 1. Site-specific recombination either integrates, deletes or reverses a DNA
                            sequence.</strong>
                        <br><br>

                    </div>

                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="p2">Aims</h2>
                        We would like to prove the feasibility of bio-flip-flops and optimize their function as parts.
                        In order to do this, we have to:<br>
                        <div class="nonumberitem">
                            <ul>
                                <li>Select effective recombinases and their corresponding RDFs; characterize and
                                    optimize
                                    their function.
                                </li>
                                <li>Construct half of the flip-flop structure and express it in <i>E. coli.</i></li>
                            </ul>
                        </div>
                        We would first search for known recombinase sequences in the literature, construct the
                        corresponding vectors, and select the best ones to make standardized parts. After confirming
                        their expression and function, we would optimize these devices through the standard workflow of
                        vector and RBS substitution. We would also prove integrase-RDF fusion protein function.
                        Furthermore, we would con-struct half of the bio-flip-flop structure and experimentally show
                        that it can function.<br><br>
                        This would serve as a proof of concept for a novel design strategy for bio-flip-flops.<br>

                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="p3">Key Achievements</h2>
                        <div class="nonumberitem">
                            <ul>
                                <li>Test and optimize recombinase and recombinase-RDF fusion protein function.</li>
                                <li>Standardization into BioBrick devices.</li>
                                <li>Introduction of the forward latch of the bio-flip-flop into cells and proof of
                                    function
                                    in vivo.
                                </li>
                            </ul>
                        </div>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="p4">Our Approach and Designs</h2>
                        <h3 id="p4.1">Design of the bio-flip-flop</h3>
                        Using a pair of integrases and their corresponding excisionases, we designed the bio-flip-flop
                        device to store state information as follows:<br>
                        <div class="nonumberitem">
                            <ul>
                                <li>The device has two promoters, controlled by input inducers X and Y, respectively.
                                    The
                                    input can either promote or inhibit transcription.
                                </li>
                                <li>In the schematic mechanism figure below, recombination sites with colors
                                    corresponding
                                    to their binding integrases are placed on both sides of the promoter in inverse
                                    directions. Thus, integrases can flip the promoters, changing the direction of
                                    transcription.
                                </li>
                                <li>We define the combination of the input series X, and then Y as a single signal.
                                    Thus,
                                    the bio-flip-flop can change the circuit state each time a signal arrives, and keep
                                    this
                                    state until the next signal pulse.
                                </li>
                            </ul>
                        </div>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/1/11/Peking_flipflop_fig2.png"
                                                 height="300px"/>
                        </div>
                        <br>

                        <strong>Figure 2. Schematic diagram of the bio-flip-flop mechanism.</strong><br>
                        <br>
                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/b/b5/Peking_flipflop_fig2_video.mp4"
                                        type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/b/b5/Peking_flipflop_fig2_video.mp4"
                                        type="video/ogg"></source>
                            </video>
                        </div>
                        <br>
                        <br>

                        <h3 id="p4.2">Selection of Available Integrases</h3>
                        We first did a literature search and sequence alignment to mine the databases for available
                        integrases for device development. Here we list the names and sources of the collected
                        recombinases.<br><br>

                        <object data="https://static.igem.org/mediawiki/parts/3/37/List_of_collected_recombinases.pdf"
                                type="application/pdf" width="100%" height="700px">
                            <p>It appears you don't have a PDF plugin for this browser.
                                No biggie... you can <a
                                        href="https://static.igem.org/mediawiki/parts/3/37/List_of_collected_recombinases.pdf">click
                                    here to
                                    download the PDF file.</a></p>
                        </object>
                        <br>

                        We finally chose to use TP901-1, Bxb1 and phiC31. These are well-known serine integrases, and
                        all of them have been used to demonstrate static-input logic gates. Some have cofactors
                        (equivalent to RDFs) that can reverse directionality. Previous work has shown that there exists
                        a new set of 11 orthogonal integrases, greatly expanding the availability of integrases that can
                        be formed into standardized parts. We hope that given enough time and resources, it is possible
                        to construct more parts using our standard workflow described below.<br>

                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="p5">Standard Characterization & Optimization Workflow</h2>
                        The viability of a bio-flip-flop relies on the performance of two integrases and their
                        corresponding excisionases. To select integrases for the bio-flip-flop, we constructed
                        expression vectors for different recombinases and tested their performance individually. Here
                        we introduce a standard workflow to accomplish this task.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/parts/7/7d/Workflow-of-flipflop.png"
                                                 height="300px"/>
                        </div>
                        <strong>Figure 3. The workflow of bio-flip-flop optimization.</strong><br><br>

                        Different replication origins affect the copy number of plasmids, and it is highly probable that
                        they also influence the expression levels of their encoded recombinases. To select integrases
                        for the bio-flip-flop, we constructed expression vectors for different recombinases and tested
                        their performance individually. This was carried out by co-transformation of <i>E. coli</i>
                        Top10 with the expression vector and a reporter plasmid. The reporter plasmid expresses differen
                        t fluorescent proteins before and after flipping of a constitutive promoter.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/f/fc/Peking_flipflop_fig_5.svg"
                                                 height="300px"/>
                        </div>
                        <br><br>
                        <strong>Figure 4. The standard genetic structure used to characterize the recombinases.</strong><br><br>

                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/1/1f/Peking_flipflop_int.mp4"
                                        type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/1/1f/Peking_flipflop_int.mp4"
                                        type="video/ogg"></source>
                            </video>
                        </div>
                        <br>

                        According to our design of the bio-flip-flop, the two inputs X and Y comprise a signal.
                        Consequently, two distinct induction systems need to be constructed. For this reason,
                        recombinases were cloned onto two different backbones, under regulation of differently induced
                        promoters. The expression vectors were constructed by Gibson assembly. Plasmid type 1 is cloned
                        from the repressor generator (RPG), which has a p15A replication origin, ampicillin resistance
                        gene, lacI, an IPTG-induced pTac promoter, and a RiboJ insulator after pTac. The backbone of
                        plasmid type 2 was cloned from pIntegrase_13, which was a gift from Christopher Voigt
                        (Addgene plasmid # 60584). It has a ColE1 replication origin, kanamycin resistance gene,
                        <i>araC</i>,
                        and an arabinose-induced pBad promoter. RiboJ was added to Plasmid2 by cloning RiboJ and the
                        recombinase from Plasmid1, and assembled after pBad.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/d/dc/Peking_flipflop_fig_6.svg"
                                                 height="400px"/>
                        </div>
                        <br>
                        <br>

                        <strong>Figure 5. Integrase expression vectors with different replication origins.</strong> The
                        one shown on top has a relaxed ColE1 replication origin and recombinase expression is induced by
                        arabinose via a pBad induction system. The one shown in the bottom picture has a relaxed p15A
                        replication origin and recombinase expression is induced by IPTG.<br><br>

                        For a given backbone and inducible promoter, RBS strength affects the expression level
                        post-transcription. For this reason, we tested a series of RBS for different recombinases in
                        different backbones.<br><br>
                        In order to tune the RBS, an intermediate vector with a 20bp random sequence (45% GC), flanked
                        by a pair of BsmbI recognition sites (introduced by PCR) was prepared for each recombinase.
                        Vector construction was completed by adding RBS via Golden Gate assembly. The RBS calculator
                        was used to generate RBS sequences with a wide range of translation initiation rates. Other
                        sequences (B0029, B0030, B0032, B0034, B0035) were obtained from the iGEM website. The RBS
                        oligos were obtained from the annealing of two complementary primers. Expression vectors for
                        each recom-binase with different RBS were tested by flow cytometry after co-transformation with
                        the reporter vec-tor. For each recombinase, an RBS correlating with low leakage (small RFP
                        subset in the cultures with no inducer) and high recombination efficiency after induction was
                        selected.<br><br>

                        <h3 id="p5.1">Measurement of Recombination Efficiency</h3>
                        We used a microplate reader to roughly measure the efficiency of the selected integrases. We
                        used flow cytometry to conduct a more accurate characterization.<br><br>

                        <h4>Microplate Readers</h4>
                        Microplate readers are instruments used to detect biological, chemical or physical events in
                        samples in microtiter plates. We used a microplate reader to detect optical density and
                        fluorescence intensity follwing the procedure as below:<br>
                        <br>

                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Pick and inoculate single colony into 1ml of LB media with antibiotics in a V-bottom
                                96-well plate. Grow the culture overnight (about 12 hours) at 37°C, 220 rpm in a Thermo
                                VARIOSKAN FLASH shaker.
                            </li>
                            <li>Then, an aliquot comprising 2 μL of the culture was transferred into 1ml LB media with
                                antibiotics and inducer (1mM IPTG or 10mM arabinose). Culture overnight (about 12 hours)
                                at 37°C and 220 rpm.
                            </li>
                            <li>Aliquot 1 μl of the cultures in the deep-well plates, then copy them to the 96-well
                                plate filled with 200μl PBS. Don’t forget to leave at least 12 wells with only PBS in
                                it as your negative conctrol.
                            </li>
                            <li>Measure the samples (OD and Fluorescence). The former represents the density of
                                bacteria,
                                and the latter implies the efficiency of recombination. GFP/OD is calculated to measure
                                recombination efficiency.
                            </li>

                        </ol>
                        <br>
                        <br>

                        <h4>Flow Cytometry</h4>
                        For more detailed measurements,flow cytometry were used to evaluate the recombination
                        efficiency.
                        Our procedures are as follows:<br>

                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Single colonies were picked and used to inoculate into 1ml of LB media with antibiotics
                                in a V-bottom 96-well plate.
                            </li>
                            <li>The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot
                                comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with
                                antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient
                                concentration for transfer curve) in a V-bottom 96-well plate.
                            </li>
                            <li>The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the
                                culture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2
                                mg/mL
                                kanamycin in a 96-well plate. This mixture was incubated for one hour at room
                                temperature
                                before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell
                                fluorescence distribution at both emission wavelengths was recorded.
                            </li>

                        </ol>
                        <br>
                        <br>

                        The counted cells were gated to eliminate the population which showed no fluorescence. The
                        remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The
                        recombination efficiency was estimated from the proportion of the RFP subset in the total
                        fluorescent population.<br>
                        <br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/ba/Peking_flipflop_fig_7.png"
                                                 height="300px"/></div>
                        <br>

                        <strong>Figure 6. A example of Gating the RFP and GFP subsets. Change of fluorescence after
                            induction was seen. Left: no inducer. Right: 10 mM Ara for 15h.</strong> Change of
                        fluorescence after induction was seen. Left: no inducer. Right: 10 mM Ara for 15h.<br><br>

                        For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs
                        for TP901-1.<br>
                        <br>

                        We observed a shift in fluorescence level and wavelength in several trails, which indicated that
                        recombinase can flip the promoter region and alter gene expression in our design. However,
                        problems such as leakage and insufficient recombination efficiency were obvious. Tuning of our
                        expression system seemed necessary before assembly into the bio-flip-flop device.<br><br>

                        <h3 id="p5.2">Characterization of Excisionases</h3>
                        To make sure that the sequence-reversing process is efficient and complete, we used two
                        strategies: one encompasses combining the integrase and its corresponding RDF into a
                        polycistronic structure, and the other is to construct fusion proteins comprising the integrases
                        and RDFs in one polypeptide chain. We have been trying to improve the recombination efficiency
                        by replacing the expression vec-tor with different replication origins or inducible promoters
                        and changing the RBS sequence before the coding sequence.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/8/8b/Peking_flipflop_fig_8.svg"
                                                 height="250px"/></div>
                        <br>
                        <strong>Figure 7. The two strategies used to characterize integrases and their RDFs.</strong>
                        UP. The polycistronic structure containing the integrase and its corresponding RDF. Bottom. The
                        fusion
                        protein structure of integrase with its corresponding RDF.<br><br>

                        The method is similar to the integrase test method. We constructed an input plasmid to express
                        integrases and RDFs, and an output plasmid on which the promoter J23119 is located between the
                        attL and attR sequences. The input plasmid expresses GFP before induction of integrase and RDF,
                        and if an inversion occurs after induction, the output plasmid can express RFP. By comparing the
                        changes in fluorescence types and intensities before and after induction, the efficiency of
                        inversion can be estimated.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/parts/9/96/RDF_diagram.png"
                                                 height="250px"/>
                            <br>
                        </div>
                        <strong>Figure 8.The standard genetic structure used to characterize the
                            recombinases.</strong><br><br>
                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/0/09/Peking_flipflop_xis.mp4"
                                        type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/0/09/Peking_flipflop_xis.mp4"
                                        type="video/ogg"></source>
                            </video>
                        </div>


                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="p6">Construction of Bio-flip-flops</h2>
                        After having already optimized the expression systems of integrases and excisionases, we set out
                        to construct a Bio-Flip-Flop. Since it is unknown whether the Bio-Flip-Flop is practical, we
                        considered developing two hierarchical execution units supporting our Bio-Flip-Flop. We called
                        these two units the forward latch and the backward latch. The state transition process of each
                        unit consists of two stable and irreversible states. Nevertheless, the state transition proces
                        s of both units consists of four stable and cyclic states. Such units support the verification
                        of the feasibility of our Bio-Flip-Flop and further allow us to construct a complete and
                        functional Bio-Flip-Flop.<br><br>

                        <h3 id="p6.1">Forward Latch</h3>
                        The structure of the forward latch consists of two plasmids with different induction systems of
                        integrases (Figure 9). The state transition process of the forward latch consists of two stable
                        and irreversible states (Figure 10).<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/8/8f/Peking_flipflop_fig_9.svg"
                                                 height="400px"/></div>
                        <br>
                        <strong>Figure 9. The structure of the forward latch.</strong> Two plasmids constitute the
                        forward latch. Each plasmid contains an integrase gene (Bxb1-gp35 or TP901) and a reporter gene
                        (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the transcription
                        of Bxb1 and GFP, and the pTAC promoter to regulate the transcription of TP901 and mRFP.
                        Moreover,
                        each integrase recognizes and converts the attB and attP sites on the other plasmid.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e3/Peking_flipflop_fig_10.svg"
                                                 height="500px"/></div>
                        <br>
                        <strong>Figure 10. The state transition process of the Forward latch.</strong> After induction
                        with arabinose, the expression of integrase Bxb1 and GFP is initiated. The integrase Bxb1
                        recognizes and converts the attB and attP sites flanking the pTAC promoter, which leads to a
                        change of orientation of the pTAC promoter. If we input IPTG next, the expression of integrase
                        TP901 and mRFP will begin. The orientation of the pBAD promoter will change. As the GFP is
                        degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.<br><br>

                        <h3 id="p6.2">Backward Latch</h3>
                        The structure of the Backward latch consists of two plasmids with different induction systems
                        driving excisionases (Figure 11). The state transition process of the Backward latch consists
                        of two stable and irreversible states (Figure 12). Because excisionases recognize attL and attR
                        sites and convert them into attB and attP sites, the backward latch is capable of resetting the
                        state of the Forward latch.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/d/de/Peking_flipflop_fig_11.svg"
                                                 height="400px"/></div>
                        <br>
                        <strong>Figure 11. The structure of the Backward latch.</strong> Two plasmids constitute the
                        Backward latch. Each plasmid contains an excisionase gene (Bxb1-gp47 or TP901) and a reporter
                        gene (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the
                        transcription of Bxb1 and GFP, and the pTAC promoter to control the transcription of TP901 and
                        mRFP. Moreover, each excisionase recognizes and converts the attL and attR sites on the other
                        plasmid.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/7/7b/Peking_flipflop_fig_12.svg"
                                                 height="600px"/></div>
                        <br>
                        <strong>Figure 12. The state transition process of the Backward latch.</strong> After induction
                        with arabinose, the expression of excisionase Bxb1 and GFP is initiated. The excisionase Bxb1
                        recognizes and converts the attL and attR sites flanking the pTAC promoter, which leads to a
                        change of the orientation of the pTAC promoter. If we input IPTG next, the expression of
                        excisionase TP901 and mRFP will begin. The ori-entation of the pBAD promoter will change. As GFP
                        is degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.<br><br>

                        <h3 id="p6.3">Experiment</h3>
                        Because our final goal is to realize the state transition of our Bio-Flip-Flop, it is
                        significant
                        to characterize the efficiency of the primary state transition of the forward latch. We
                        quantified the amounts of GFP and mRFP and considered the ratio of mRFP/GFP fluorescence as a
                        direct measure of the efficiency of the state transition.<br><br>
                        For all characterization experiments, co-transformed cells were first grown and subcultured
                        overnight, after which we induced the cells with arabinose and IPTG in that order. Finally, we
                        quantified the fluorescence strength using a flow cytometer.<br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="p7">Results</h2>

                        <h3 id="p7.2">Primary Proof of Recombination</h3>

                        To prove the function of our integrase-RDF fusion protein in an illustrative way, IPTG was
                        applied to the petri dish
                        where E. coli Top10 co-transferred with expression and reporter plasmid were cultured. Compared
                        to the RFP control (transferred with RFP expressing reporter plasmid only), green fluorescence
                        was obvious when excited by visible light. Half red, half green colonies were seen, indicate
                        incomplete recombination.<br><br>

                        <div align="center">
                            <img src="https://static.igem.org/mediawiki/2017/f/fb/Tp901-gfp-control-green.jpg"
                                 height="180px"/>
                            <img src="https://static.igem.org/mediawiki/2017/9/9b/Tp901-rdf-flip.png" height="180px"/>
                            <img src="https://static.igem.org/mediawiki/2017/7/7a/Peking_Tp901-rdf-flip-close.jpg"
                                 height="180px"/>
                        </div>
                        <br>
                        <strong>Figure 13.</strong> (left) RFP control. (middle) expression vector of TP901-RDF fusion
                        protein co-transferred with
                        reporter. White arrows: colonies with green fluorescence indicates recombination. (right) A
                        example of half red, half green colonies, indicating incomplete recombination.<br>
                        <br>

                        In a similar trail, Arabinose was applied to the petri dish where E. coli Top10 co-transferred
                        with TP901-1 integrase expression and reporter plasmid were cultured. Compared to the GFP
                        control (transferred with GFP expressing reporter plasmid and expression vector, but no inducer
                        was added), We can see a transition of colony color from green (GFP expressin) to red (RFP
                        expression). When excited by visible light, it’s distinguishable that the colonies nearly
                        completely cease to express GFP. The expression of RFP and shut down of GFP expression shows
                        that recombination has happened.<br><br>

                        <div align="center">
                            <img src="https://static.igem.org/mediawiki/2017/e/e0/Resite-rfp-control.jpeg" height="200px"/>
                            <img src="https://static.igem.org/mediawiki/2017/5/54/Peking_Tp901.jpeg" height="200px"/>
                            <br>
                            <img src="https://static.igem.org/mediawiki/2017/d/d8/Peking_Tp901-flip.jpeg" height="200px"/>
                            <img src="https://static.igem.org/mediawiki/2017/f/f9/Peking_Tp901-gfp-control.jpeg"
                                 height="200px"/>
                        </div>
                        <br>
                        <strong>Figure 14.</strong> (Up, left) GFP control, colonies in green. (Up, right) GFP control,
                        green
                        fluorescence can be seen when excited. (Bottom, left) Expression vector of TP901-1 integrase
                        co-transferred with reporter, red color instead of green indicates recombination. (Bottom,
                        right) Expression vector of TP901-1 integrase co-transferred with reporter, green fluorescence
                        is nearly completely shut down, indicating desirable recombination efficiency.<br>
                        <br>

                        <h3 id="p7.1">Fine-tuning of Recombination Efficiency</h3>

                        <h4>Selection of RBS for different replication origin</h4>
                        For the vector with ColE1 replication origin, we found proper RBS in a list of
                        calculated RBSs for TP901-1 and Bxb1 gp35.<br><br>


                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/2/2c/Peking_flipflop_fig14.png"
                                                 height="500px"/>
                        </div>
                        <br>
                        <strong>Figure 15. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS
                            sequences from iGEM (B0030&sim;B0035).</strong> <br> T.I.R = Translation Initiation Rate<br><br>


                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/b8/Peking_Flip_flop_Fig16A.png"
                                                 height="500px"/>
                        </div>
                        <br>
                        <strong>Figure 16. TP901-1 recombination efficiency with a variety of calculated RBS.</strong>
                        A. At the vector with p15A. <br> B. At the vector with colE1. <br> T.I.R = Translation
                        Initiation Rate <br><br>

                        For the expression vector with p15A replication origin, a proper RBS for TP901-1 was
                        selected.<br><br>

                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/parts/5/54/TP901-1_Flipping_Efficiency_p15A.png"
                                height="500px"/>
                        </div>
                        <br>
                        <strong>Figure 16_b. TP901-1 recombination efficiency with a variety of RBS from iGEM (B0030&sim;B0035).</strong> <br>
                        T.I.R = Translation Initiation Rate<br><br>

                        <h4>Transfer curves</h4>
                        We evaluated the performance of phiC31 and Bxb1 gp35 on ColE1 expression vectors under a series
                        of inducer concentration gradients. The results enabled us to determine the appropriate inducer
                        concentration.<br><br>

                        <div align="center">
                            <strong>A</strong>
                            <img src="https://static.igem.org/mediawiki/2017/7/7d/Peking_flipflop_fig16.png" height="500px"/>
                            <strong>B</strong>

                            <img src="https://static.igem.org/mediawiki/2017/9/93/Peking_flipflop_fig17.png" height="500px"/>
                        </div>
                        <br>
                        <strong>Figure 17. The transfer curves of the recombinases we utilized.</strong> A. The transfer
                        curve of integrase phiC31. B. The transfer curve of integrase Bxb1.<br><br>


                        <h4>Measurement of the Flipping Efficiency of Excisionases</h4>
                        We have tested the flipping efficiency of our Integrase-RDF fusion proteins with a group pf RBS.
                        Flipping performance varies with different RBS. For TP901-RDF fusion proteins, recombination
                        seems not obvious in some cases (RBS B0032, B0034, B0035, phiC 10000, Bxb 1000, int2 3000),
                        while in others both flipping efficiency and leakage and significant.<br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/c/c6/Peking_filpflop_fig_17.png"
                                                 height="600px"/>
                        </div>
                        <br>

                        <strong>Figure 18. pTac TP901-RDF fusion protein flipping efficiency with ten RBS.</strong>
                        Proportion of GFP subset indicates flipping efficiency.<br><br>

                        A second test was supplemented. Similar results support that our Integrase-RDF fusion proteins
                        are able to flip the sequence between attL and attR site of the reporter, but much improvement
                        are needed to reduce the leakage as well as increase the efficiency.<br>
                        <br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/c/ca/Peking_filpflop_fig_18.png"
                                                 height="600px"/>
                        </div>
                        <br>

                        <strong>Figure 19. pTac TP901-RDF fusion protein flipping efficiency with two RBS.</strong>
                        Proportion of GFP subset indicates flipping efficiency.<br><br>

                        Integrase-RDF for Bxb1 and phiC31 were also constructed, tested. High flipping efficiency and
                        leakage were seen.<br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/c/c4/Peking_filpflop_fig_19.png"
                                                 height="300px"/>
                        </div>
                        <br>

                        <strong>Figure 20. Flipping efficiency for pTac integrase-RDF fusion protein of phiC31 and
                            Bxb1.</strong>
                        Proportion of GFP subset indicates flipping efficiency.<br><br>

                        To conclude, we found that the Integrase-RDF fusion proteins are able to flip the sequence
                        between attL and attR site (indicated by observed GFP subset), although tuning work remains to
                        be done to construct a controllable, functional bio-flip-flop device.<br>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="p8">Reference</h2>
                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Yang, L., Nielsen, A. A., Fernandez-Rodriguez, J., McClune, C. J., Laub, M. T., Lu, T.
                                K., & Voigt, C. A. (2014). Permanent genetic memory with> 1-byte capacity. Nature
                                methods, 11(12), 1261-1266.
                            </li>
                            <li>Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., & Endy, D. (2013). Amplifying
                                genetic logic gates. Science, 340(6132), 599-603.
                            </li>
                            <li>Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (1970). Molecular biology
                                of the gene.
                            </li>
                            <li>Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C., Colloms, S. D., &
                                Stark, W. M. (2017). Control of serine integrase recombination directionality by fusion
                                with the directionality factor. Nucleic acids research, 45(14), 8635-8645.
                            </li>
                            <li>Siuti, P., Yazbek, J., & Lu, T. K. (2013). Synthetic circuits integrating logic and
                                memory in living cells. Nature biotechnology, 31(5), 448-452.
                            </li>
                            <li>Khaleel, T., Younger, E., McEwan, A. R., Varghese, A. S., & Smith, M. (2011). A phage
                                protein that binds φC31 integrase to switch its directionality. Molecular microbiology,
                                80(6), 1450-1463.
                            </li>
                            <li>Salis, H. M., Mirsky, E. A., & Voigt, C. A. (2009). Automated design of synthetic
                                ribosome binding sites to control protein expression. Nature biotechnology, 27(10),
                                946-950.
                            </li>
                            <li>Espah Borujeni, A., Channarasappa, A. S., & Salis, H. M. (2013). Translation rate is
                                controlled by coupled trade-offs between site accessibility, selective RNA unfolding and
                                sliding at upstream standby sites. Nucleic acids research, 42(4), 2646-2659.
                            </li>

                        </ol>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp" id="Tflipflop">

                    <div class="mdl-card__supporting-text">

                        <h2>T Flip-Flop</h2>
                        Basically, a real flip flop has two inputs. One input is a control input. For a T flip flop, the
                        control input is labelled T. The other input is the clock. Positive edge-triggered flip flops
                        can only change output values when the clock is at a positive edge. While negative edge
                        triggered flip flops change on a negative edge, and level-triggered flip flops, that change only
                        when the value is 1. When the clock is not at a positive edge, then the output value is held.
                        That is, it does not change. A flip flop’s output is the bit it stored. Thus, the flip flop is
                        always outputting the one bit of information.<br>
                        Here's the characteristic table for a T flip flop.<br><br>

                        <table class="mdl-data-table mdl-js-data-table">
                            <thead>
                            <tr>
                                <th class="mdl-data-table__cell--non-numeric">T</th>
                                <th>Q</th>
                                <th>Q+</th>
                                <th>Operation</th>
                            </tr>
                            </thead>
                            <tbody>
                            <tr>
                                <td class="mdl-data-table__cell--non-numeric">0</td>
                                <td>0</td>
                                <td>0</td>
                                <td>Hold</td>
                            </tr>
                            <tr>
                                <td class="mdl-data-table__cell--non-numeric">0</td>
                                <td>1</td>
                                <td>1</td>
                                <td>Hold</td>
                            </tr>
                            <tr>
                                <td class="mdl-data-table__cell--non-numeric">1</td>
                                <td>0</td>
                                <td>1</td>
                                <td>Toggle</td>
                            </tr>
                            <tr>
                                <td>1</td>
                                <td>1</td>
                                <td>0</td>
                                <td>Tohgle</td>
                            </tr>
                            </tbody>
                        </table>
                        <br>
                        <br>

                        The T flip flop characteristic table has 3 columns. The first column is the value of T, a
                        control input. The second column is the current state, that is the current value being output by
                        Q. The third column is the next state, that is, the value of Q at the next positive edge. It's
                        labelled with Q and the superscript, + (the plus sign).<br>
                        <br>
                        The T flip flop has two possible values. When T = 0, the flip flop does a hold. A hold means
                        that the output, Q is kept the same. When T = 1, the flip flop does a toggle, which means the
                        output Q is negated. Thus, in a T flip flop, you can either maintain the current state's value
                        for another cycle, or you can toggle the value (negate it).<br>
                        <br>
                        <strong>Why isn't it XOR?</strong><br>
                        First, XOR has two inputs, and one output. A T flip flop essentially has a control input and a
                        control output. Second, the second column and the third column are really the same output, but
                        at different points in time.<br>
                        <br>
                        <strong>Difference between Flip flop and gate</strong><br>
                        The biggest difference between a flip flop and a gate is that a flip flop can hold its value.
                        Even though holding a value is something very simple, it makes it different from logic gates,
                        and allows us to design circuits that have cycles in them (i.e., feedback).<br><br>
                    </div>

                </div>

                <div id="State" class="demo-card-wide mdl-card mdl-shadow--2dp">

                    <div class="mdl-card__supporting-text">

                        <h2>State</h2>
                        There is a generic definition of state:<br>
                        <i>State is a quantity that stores the previous history of what's happened in such a way that
                            you can predict the output, given any future input.</i>
                        <br><br>
                        From the definition, we can get two things. First, state refers to some recorded information. We
                        can consider state as a kind of memory. For sequential logic circuits, this is some finite
                        number of bits. Second, there is always a notion of time when we talk about state. Usually, we
                        think of time being involved with state, because it may change over time.<br>

                        Let’s think of an object, it starts off in an initial state, and over time, that state changes.
                        For example, if the object is a piece of paper, when we are drawing we may draw or erase
                        something (add or remove elements) over time. Our picture’s state may not keep track of all the
                        things that have happened to it, nevertheless, the picture changes from state to state, as
                        various operations are performing object.<br>

                        <br><a href="https://static.igem.org/mediawiki/2017/0/0f/Peking_flipflop_Asynchronous_Counters.pdf"
                               target="blank" style="color: #226CE4"><strong>Learn More</strong></a>

                        <br><br><a
                            class="mdl-button mdl-button--accent mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                            href="#top" style="color: white;">
                        Back to top
                    </a>

                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->

                </div>

            </section>
            <section id="Controller" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">

                    <div class="mdl-card__supporting-text">

                        <h1>Controller</h1>
                        <br>


                        <section class="docs-toc docs-text-styling" style="margin-left: 0px">
                            <nav class="section-content" style="margin-left: 0px">
                                <ul style="margin-left: 0px">

                                    <li><a href="#pc0">Background</a></li>
                                    <li><a href="#pc1">Aim</a></li>
                                    <li><a href="#pc2">Key Achievements</a></li>
                                    <li><a href="#pc3">Our Approach and Designs</a></li>
                                    <li><a href="#pc5">Results</a></li>
                                    <li><a href="#pc6">References</a></li>

                                </ul>
                            </nav>
                        </section>

                        <h2 id="pc0">Background</h2>
                        In our design, after having designed the flip flop, the device can remember the information of
                        its state, the next step is to transform the state into an actual function. To achieve this
                        transformation, we first needed a “reader” to read out the current state. At this point the
                        control unit comes into play. A control unit is a DNA sequence with recombinase sites whose
                        expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a
                        terminator. To make the control unit reliable and predictable, we first need to be able to
                        predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from
                        which we weave our engineer’s perspective into the biolog-ical system. However, we need to make
                        some adaptations and adjustments to these “elements” to make them usable.
                        <br><br>

                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/f/f0/Peking_controller_terminator.mp4"
                                        type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/f/f0/Peking_controller_terminator.mp4"
                                        type="video/ogg"></source>
                            </video>
                        </div>


                    </div>

                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="pc1">Aim</h2>
                        <div class="nonumberitem">
                            <ul style="line-height: 2em;">
                                <li>To characterize a group of terminators and measure their strength in both
                                    orientations.
                                </li>
                                <li>To select unidirectional terminators that are able to serve as switches in genetic
                                    circuits.
                                </li>
                                <li>To measure the strength of terminators flanked by one set of attB/P sites, and the
                                    inversion efficiency of recombinases.
                                </li>
                                <li>To measure the terminator strength of terminators flanked by two sets of attB/P
                                    sites,
                                    and the inversion efficiency of different recombinases.
                                </li>
                                <li>To construct a 7-segment display which is able to count from 1 to 3.</li>
                            </ul>
                        </div>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="pc2">Key Achievements</h2>
                        <div class="nonumberitem">
                            <ul style="line-height: 2em;">
                                <li>In our project, after we have made these constructive modifications to terminators
                                    and
                                    recombinases to meet our requirements of robust, reliable and predictable behaviors,
                                    which will be presented in the form of a seven-segment display. This will improve
                                    our
                                    quantitative understanding of natural phenomena and foster an engineering discipline
                                    for obtaining new complex cell behaviors.
                                </li>
                                <li>We measured the strengths of a total of 24 terminators in both directions, and we
                                    elected 6 suitable unidirectional terminators.
                                </li>
                                <li>We measured the strengths of several terminators paired with attB/P sites and the
                                    inversion efficiency of 3 recombinases.
                                </li>
                                <li>We succeeded to construct the transformation of the pattern from ”2017” to ”PKU”.
                                </li>
                            </ul>
                        </div>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="pc3">Our Approach and Designs</h2>

                        <h3 id="pc3.1">Characterization of Terminators</h3>
                        Terminators are responsible for disengaging the RNA polymerase from the DNA and stopping gene
                        transcription in vivo. This characteristic makes terminators desirable as switches in synthetic
                        genetic circuits. The terminators that are used to realize this function should be
                        unidirectional.
                        That is, they should be able to block the movement of RNA polymerase effectively in one
                        orientation and hardly influence its movement in the other. With this “reader”, advantages
                        include a shorter sequence, per-manently recorded message in the DNA and greater number of
                        design plans. Consequently, it is es-sential that we characterize the different terminators.
                        We first chose 24 terminators whose forward orientation strength is high, as previously
                        reported.
                        We then measured their strength in both the forward and reverse orientations in our testing
                        system.
                        Finally, we sifted out a set of six reliable unidirectional terminators without potential
                        cryptic promoters in both orientations.<br>
                        <br>
                        Given that the same part can perform differently in different genetic circuits, previous testing
                        systems and results of terminators may not fit our project. We therefore constructed a rather
                        simple but practical reporter plasmid (as shown below), based on which terminator strength can
                        be measured via fluorescence intensity and simple calculations.<br>
                        <br>

                        <h4>Vector construction</h4>
                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/6/6f/Peking_controller_figure1.svg"
                                height="220px"/></div>
                        <br>
                        <strong> Figure 1. Vector construction to measure the terminator strength</strong><br><br>


                        As shown above (Figure 1.), the circuit used to measure terminator strength was constructed on
                        the pSB4C5 vector. The terminator to be tested is located downstream of the Ptac promoter and
                        followed successively by RiboJ, RBS, and sfGFP. To enable high throughput, we introduced a pair
                        of BsaI cutting sites between Ptac and RiboJ so that Golden Gate assembly can be used to add
                        different terminators efficiently. In addition, there is a random sequence (from a previous
                        associated research, the author of which had designed it using the Sequence Manipulation Suite)
                        between the two Bsal sites flanked by two RiboJ, which was constructed as a positive
                        control.<br>
                        The terminators to be tested were selected from the literature.<br>
                        <br>
                        RiboJ acts as an insulator between the RBS and its upstream sequence in the genetic circuit. The
                        insulating function of RiboJ is directly associated with its biochemical function. RiboJ can be
                        defined as the DNA sequence containing a ribozyme called sTRSV- ribozyme and a 23-nt hairpin.
                        The downstream hairpin helps to expose the RBS. mRNA can be cleaved by sTRSV autocatalytically
                        at a defined residue. As a result, extraneous RNA leaders that arise from transcription from
                        promoters with different start sites will be cleaved. Moreover, to make sure the sequence
                        upstream of the RBS will not affect RiboJ’s efficiency, the transcripts of our genetic circuits
                        were designed to have the same 5’-UTR sequence, to avoid the effect called “part junction
                        interference”, in which neighboring sequences may alter the part’s character. For example, a
                        barcode part was found to contain a sequence similar to the –10 region of a constitutive
                        promoter. <br>
                        <br>

                        <h4>Flow Cytometry</h4>
                        Vectors constructed with different terminators in both directions were tested via flow
                        cytometry.<br>
                        <i>E. coli </i> strains were grown for 12h in LB media containing antibiotics in culture tubes
                        at 37°C and 250 rpm in an incubator. After 12h, the cultures were diluted 1:100 with M9 medium
                        with antibiotics in 96-deep-well plates and grown for 2h at 37°C and 1150rpm in a shaker. Next,
                        the cultures were diluted 1:100 with fresh M9 medium with antibiotics and different
                        concentrations of IPTG. These cultures were grown for 6 h and then diluted 1:5 with
                        phosphate-buffered saline (PBS) containing 2 mg/ml kanamycin or 35 ug/ml chloramphenicol to
                        arrest protein production, and fluorescence was measured using a flow cytometry.<br>
                        <br>
                        Cells were analyzed by flow cytometry using a BD biosciences Fortessa flow cytometer with blue
                        (488-nm) laser. An injection volume of 15 μl and a flow rate of 1 μl/s were used. Cytometry data
                        were analyzed using Flowjo. The gated populations consisted of at least 10 000 cells. The median
                        fluorescence of the gated populations was calculated using Fowjo and used for all reporting.
                        Autofluorescence of white cells was subtracted from all fluorescence measurements.<br><br>
                        <h4>The Mathematical Formula</h4>
                        As shown below, a mathematical formula was also used to characterize the strength of the
                        terminators. ＂Ts＂ is the abbreviation of ＂terminator strength＂. [GFP]<sub>random sequence</sub>
                        denotes the fluorescence strength of the plasmid with the random sequence.
                        [GFP]<sub>terminator</sub> denotes the fluorescence strength of the plasmid with a
                        terminator.<br><br>

                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/f/fb/Peking_controller_equation1.png"
                                height="100px"/>
                        </div>

                        Ideal unidirectional terminators should fit the following standards:<br>

                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>The Ts of both directions are larger than 0.9 (i.e. there is no cryptic promoter
                                potential)
                            </li>
                            <li>The Ts ratios (Ts of the forward direction/Ts of the reverse direction) are larger than
                                10
                            </li>
                            <li>The larger Ts between the forward and reverse directions has to be larger than 10 (which
                                ensures the blocking strength to be high enough)
                            </li>
                            <li>The smaller Ts between the forward and reverse directions has to be no larger than 1.2
                                (i.e. terminators in reverse orientation no longer block the transcription process).
                            </li>
                        </ol>
                        <br><br>

                        <h4>To remeasure the Ts of the unidirectional terminators</h4>
                        After the preliminary sifting of the terminators, a set of desirable unidirectional terminators
                        were sifted out and re-measured using the plate reader to refine the parameters.<br><br>
                        TOP10 cells were co-transformed with the system containing the terminator and one pair of attB/P
                        sites and the expressing system. Cells then were cultured overnight on LB plates.<br><br>
                        Colonies were picked and cultured in liquid media with antibiotics and inducer. The bacterial
                        liquid culture was then diluted at the ratio of 1:100 into M9 media with both antibiotics and
                        inducer into 96-well plate, cultured for 12-24 hours in the incubator. After the induction,
                        OD600 and fluorescence were measured using the plate reader. Terminator strength was then
                        calculated according to the formula above.<br><br>


                        <h3 id="pc3.2">Characterization of Inversion Efficiency</h3>
                        After finishing these measurements, the main protagonist of our project, the recombinase, will
                        come into play. When terminators are flanked by a pair of attB/P sites, the synthetic circuit
                        will transit between on and off state, which is achieved by controlling the expression of the
                        respective recombinases. Here the question becomes whether the attB/P sites flanking the
                        terminators may alter their strength due to some unknown interaction. Thus, we needed to
                        remeasure their strength. Additionally, the inversion efficiency of respective recombinases
                        need to be characterized.<br>
                        <br>
                        We introduced the testing system and expression system of the corresponding recombinases into
                        the same cell to see if inversions happened, and if leaky expression of recombinases would
                        impact the system. In the end, couples of terminators and recombinases with near-complete
                        inversions and minimal leakage were selected.<br>
                        <br>

                        <h4>Vector construction</h4>
                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/0/08/Peking_controller_figure2.svg"
                                height="220px"/></div>
                        <strong>Figure 2. Terminator flanked by a pair of attB/P sites</strong><br>
                        <br>
                        As shown above, we selected terminators for both directions that were sandwiched between
                        attB/attP recognition sites, on the basis of the aforementioned terminator strength testing
                        system. The constructed terminators are shown in the table below.<br><br>
                        <div align="center">
                            <table class="mdl-data-table mdl-js-data-table">
                                <thead>
                                <tr>
                                    <th class="mdl-data-table__cell--non-numeric">Recombinase Recognition Site
                                        (attB/attP)
                                    </th>
                                    <th>Terminators</th>
                                </tr>
                                </thead>
                                <tbody>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">TP901-1(AG)</td>
                                    <td>855 F/R,836 F/R,435 F/R,309R,221 F/R</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">TP901-1(TC)</td>
                                    <td>836 F/R,435 F/R,322R</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Bxb1 (GT)</td>
                                    <td>836 F/R,435 F/R,322F/R</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Int2</td>
                                    <td>855 F/R,836 F,435 FR,322 R</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">ΦC31</td>
                                    <td>855 F/R,836 F/R,435 F/R,309 R,221 FR</td>
                                </tr>
                                </tbody>
                            </table>
                        </div>
                        <br>
                        <strong>Those letters in pairs inside the brackets stand for different two-base core inside the
                            sequence of recombinase recognition sites, which remained unchanged after
                            inversion.</strong>
                        The expression system includes the recombinase Φc31, Bxb1, TP901-1, or int2 under the control of
                        the inducible pBAD promoter and optimal RBS, which can simultaneously ensure high efficiency and
                        low leakage.<br><br>
                        As a part working in genetic circuits, the terminator flanked with a pair of att B/P sites is a
                        brand new terminator in a sense. It’s necessary for us developers to measure the Ts of the new
                        terminator.<br><br>
                        <h4>Remeasurement of Ts of the new terminators</h4>
                        The plate reader is which we use to remeasure. The <a
                            href="https://2017.igem.org/Team:Peking/Protocols"
                            target="blank"
                            style="color: #226CE4"><strong>protocol</strong></a> and the formula are same as the page
                        above.
                        <br><br>
                        <h4>Characterization of inversion efficiency</h4>
                        <strong>Activation and induction</strong>
                        <i>Escherichia coli</i> colonies were picked and grown for 12h in LB media containing
                        antibiotics in culture tubes at 37°C and 250 rpm in an incubator. Next, the bacterial cultures
                        were diluted at a ratio of 1:100 with fresh M9 medium with antibiotics and inducers, after which
                        the bacteria were grown for another 12h.<br><br>
                        <strong>Plate reader</strong>
                        Fluorescence was measured using Varioskan© Flash. We measured the OD600 and green fluorescence
                        (the excitation wave length is 485nm and the emission wave length is 510nm). The ratio of
                        fluorescence intensity/OD600 was calculated using the net fluorescence intensity and net OD600.
                        The transcription barrier strength of an attB/P-Terminator can be characterized quantitatively
                        using the Ts formula.<br><br>
                        The inversion efficiency was calculated according to the below formula.<br><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/parts/1/13/Inversion_formula.png"
                                                 height="100px"/>
                        </div>

                        F<sub>ter</sub>stands for the fluorescence intensity of the system containing both the
                        terminator and the recombinase elements.<br>
                        F<sub>control</sub> stands for the fluorescence intensity of the system containing only the
                        terminator element.<br>
                        F<sub>forward</sub> stands for the fluorescence intensity of the system containing both the
                        forward terminator and the recombinase elements.<br>
                        F<sub>reverse</sub> stands for the fluorescence intensity of the system containing both the
                        reverse terminator and the recombinase elements.<br>

                        The difference between the fluorescence intensity of the system expressing a recombinase and the
                        system not with recombinase expression, divided by the difference between the fluorescence
                        intensity of the system containing the terminator in the forward direction and the system
                        containing the terminator in the reverse direction.<br><br>

                        <h3>Characterization of Terminator Flanked with Two Pairs of attBP Sites</h3>
                        <strong>Vector Construction</strong>
                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/e/ec/Peking_controller_figure3.svg"
                                height="300px"/>
                        </div>
                        <strong>Figure 3. Terminator flanked by two pairs of attB/P sites</strong><br><br>
                        As shown above(Figure 3.), the terminator is sandwiched between two different pairs of attB/attP
                        sites, under the control of a constitutive J23119 promoter.<br><br>
                        Two sets of <a
                            href="https://static.igem.org/mediawiki/2017/d/dc/Peking_flipflop_fig_6.svg"
                            target="blank"
                            style="color: #226CE4"><strong>expression systems</strong></a>, induced by IPTG and
                        arabinose, were constructed. Same as before, a mathematical formula was also used to
                        characterize the strength of the terminators. [GFP]<sub>random sequence</sub> denotes the
                        fluorescence strength of the plasmid with the random sequence. [GFP]<sub>terminator</sub>
                        denotes the fluorescence strength of the plasmid with a terminator.
                        <div align="center"><img src="https://static.igem.org/mediawiki/parts/9/9b/Ts_formula.png"
                                                 height="100px"/>
                        </div>
                        The inversion efficiency calculation formula is same as above.<br><br>

                        <h3>Construction of Seven-Segment Display</h3>
                        After constructing two reliable elements with predictable behaviors, we aimed to directly
                        implement and arrange them according to sequential logic, which comes in the form of a visible
                        “1→2→3” transition on the seven-segment display. In this display, GFP expression is used instead
                        of an LCD (liquid crystal display) to light up the panel. In our case, three simple and
                        previously characterized parts were placed in segments except F. The system yields the
                        predictable expression of recombinases which invert terminators between corresponding attB/attP
                        sites either once (A,D,G) or twice (C,E).<br><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/c/c5/Peking_pattern_1.png"
                                                 height="450px"/>
                        </div>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/8/8d/Peking_pattern_2.png"
                                                 height="450px"/>
                        </div>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/8/8e/Peking_pattern_3.png"
                                                 height="450px"/>
                        </div>
                        <strong>Figure 4. The schematic of Seven-segment Display</strong><br>

                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/5/52/Peking_controller_123.mp4"
                                        type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/5/52/Peking_controller_123.mp4"
                                        type="video/ogg"></source>
                            </video>
                        </div>

                        <h3>Construction of pattern“2017”→“PKU”in 96-well plate</h3>
                        Cells were placed according to the pattern of “2017”and “PKU”, and cultured in the 96-deep-well
                        plate. Then cells were transferred into clear-96-well plate, OD600 and fluorescence were
                        measured using the plate reader. The transition from the pattern of “2017” to “PKU” was achieved
                        using the inversion effect.<br><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/d/d2/Peking_pattern_PKU.png"
                                                 height="300px"/>
                        </div>
                        <br>
                        <strong>Figure 5. The schematic of “2017”→“PKU”</strong><br><br>
                        The holes marked “a” is always light. The ones marked “b” need to transform from the light state
                        to the dark state. And the ones marked “c” perform in contrary.<br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <h2 id="pc5">Results</h2>

                            <h3 id="pc5.1">Characterization of Terminators</h3>
                            A total of 24 terminators were tested in both orientations (their code names[1] and
                            abbreviations are shown in the table below). And a gradient of different concentrations of
                            IPTG (0, 0.01, 0.1, 1mM) was used for induction. We found that the calculated Ts values
                            varied
                            with IPTG concentration. Commonly, the Ts value for the forward orientation increased with
                            IPTG concentration and reached the highest value at 0.1mM, after which it declined slightly.
                            In the reverse orientation there were no obvious trends. Consequently, 0.1mM of IPTG was the
                            most suitable concentration for induction. After the test, we chose 6 terminators
                            (221,836,435,
                            322,855 and 309), among which 435 and 322 showed the best performance and conformed best to
                            the
                            4 standards described above.<br><br>

                            <object data="https://static.igem.org/mediawiki/2017/e/ee/Peking_controller_table2.pdf"
                                    type="application/pdf" width="100%" height="700px">
                                <p>It appears you don't have a PDF plugin for this browser.
                                    No biggie... you can <a
                                            href="https://static.igem.org/mediawiki/2017/e/ee/Peking_controller_table2.pdf">click
                                        here to
                                        download the PDF file.</a></p>
                            </object>

                            <br>
                            (The code names and their abbreviations.)<br><br>

                            <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/b0/Ts_0_1mM.png"
                                                     height="500px"/>
                            </div>
                            <br>

                            <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e3/Unidirectional_1mM.png"
                                                     height="500px"/>
                            </div>
                            <br>

                            <div align="center"><img
                                    src="https://static.igem.org/mediawiki/2017/6/65/Unidirectional_0_1mM.png"
                                    height="600px"/>
                            </div>
                            <br>

                            <div align="center"><img
                                    src="https://static.igem.org/mediawiki/2017/9/90/Peking_good_terminators.png"
                                    height="600px"/>
                            </div>
                            <br>

                            <br><br>


                            <h3 id="pc5.2">Characterization of Inversion Efficiency</h3>
                            <strong>Ts of the different attB/P+terminator combinations</strong><br>
                            Transformation with the testing system plasmid alone yielded the following results and
                            conclusions:<br>
                            <br>

                            Sole attB/P sites (without a terminator) can block the transcription to a certain extent.
                            Such barrier strength varied with the types of the sites. Among the 5 pairs of attB/P sites
                            tested, int2 had the strongest intrinsic barrier effect ("Ts"= 23.0), even exceeding that of
                            the terminator 221F ("Ts"=16.6). The other sites had weaker barrier effects: int2＞ΦC31
                            ("Ts"=2.00)＞TP901-1(AG)("Ts"=1.42).<br>
                            <br>

                            <h4>Combination of different attB/P sites and terminators can produce unexpected
                                results</h4>
                            Int2 attB/P and 435F had the strongest barrier strengths among the recognition sites and
                            terminators, respectively. However, the barrier strength of their combination was the
                            weakest
                            (“Ts”=5.37) among all combinations.<br>
                            TP901-1(AG), attB/P and 221F had the weakest barrier strength among recognition sites and
                            terminators, respectively, but the barrier strength of their combination was the
                            highest.<br>
                            In addition to the barrier effect, the combination of attB/P sites and terminators can also
                            lead to the formation of cryptic promoters, which was the case for the TP901-1(AG) 221R
                            combination.<br>
                            <br>
                            The most excellent combinations of attB/P sites and terminators selected based on the four
                            standards above were (with the corresponding “Ts” ratios in brackets): int2 221 (35.3), Φc31
                            435 (28.3), TPAG 221 (17.7), TPAG 435 (17.3), int2 855 (17.2), and BxGT435 (21.1).<br>
                            Considering that the expression systems of int2 and TP901-1 are induced by IPTG, which
                            causes a
                            conflict with the reporting system, we used BxGT435 and Φc31 435, whose systems are induced
                            by
                            Ara, to conduct the reversal experiments.<br>

                            <div align="center"><img src="https://static.igem.org/mediawiki/2017/a/a2/2nd_generation_Ts.png"
                                                     height="600px"/>
                            </div>
                            <br>
                            <br>

                            <h4>Reversal Experiment</h4>
                            <strong>Co-transformation of E. coli with the testing system plasmid and corresponding
                                recombinase expression system </strong>
                            We observed successful reversal events after inducing the expression of the recombinase. As
                            shown in the picture of the 96-well plate, the fluorescence difference is visible by naked
                            eye.
                            Using the microplate reader and analysis, we confirmed the existence of a significant
                            fluorescence difference. In addition, we directly used the bacterial solution to conduct a
                            PCR,
                            and the sequencing results showed double peaks, which substantially proves that the reversal
                            occurred.<br>
                            <br>
                            <div align="center"><img
                                    src="https://static.igem.org/mediawiki/2017/6/6a/Peking_controller_figure9.png"
                                    height="750px"/>
                            </div>
                            <br>
                            <br>
                            <div align="center"><img
                                    src="https://static.igem.org/mediawiki/2017/0/00/Peking_controller_figure10.png"
                                    height="600px"/>
                            </div>
                            <br>
                            <br>

                            <h3 id="pc5.3">Characterization of Terminators Flanked by Two Pairs of attB/P Sites</h3>
                            <strong>We built the following constructs:</strong><br><br>


                            <object data="https://static.igem.org/mediawiki/2017/f/fb/Peking_controller_table3.pdf"
                                    type="application/pdf" width="100%" height="700px">
                                <p>It appears you don't have a PDF plugin for this browser.
                                    No biggie... you can <a
                                            href="https://static.igem.org/mediawiki/2017/f/fb/Peking_controller_table3.pdf">click
                                        here to
                                        download the PDF file.</a></p>
                            </object>
                            <br>

                            Two sets of expression systems, induced by IPTG and arabinose, were constructed.<br>
                            <br>


                            <h4>Ts of double attB/P+terminator combinations</h4><br>

                            <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/b1/3rd_generation_Ts.png"
                                                     height="300px"/>
                            </div>
                            <br>

                            Due to the introduction of double attB/P sites, which substantially extended the distance
                            from
                            the promoter to the GFP coding sequence, together with the barrier effect of each pair of
                            attB/P
                            sites, it became impractical to expect the Ts of the attB/P with a reverse terminator to be
                            around 1. We there-fore lowered the standard and laid more emphasis on the ratio of Ts for
                            both
                            orientations. Based on the test results above, we chose <strong>TP901-1 BxGT435</strong> and
                            <strong>Φc31 BxbGT 435</strong> to
                            conduct the reversal experiment.<br>
                            <br>

                            <h4>Reversal Experiment</h4>
                            <div align="center"><img
                                    src="https://static.igem.org/mediawiki/2017/4/4f/3rd_generation_once_inversion.png"
                                    height="500px"/>
                            </div>
                            <br>
                            <br>


                            <h3>Image Transition Demos</h3>
                            The <i>E. coli</i> groups with “Bxb1 T+B435F” were placed at the position c, and the <i>E.
                            coli</i> groups with “Bxb1 T+B435R” were placed at the position b. The a position there are
                            groups which always expressed sfGFP.<br><br>
                            The <i>E. coli</i> colonies were picked and grown for 12h in LB media containing antibiotics
                            in culture tubes at 37°C and 250 rpm in an incubator. Next, the bacterial cultures were
                            diluted at a ratio of 1:100 with fresh M9 medium with antibiotics and without inducers,
                            after which the bacteria were grown for another 12h. We measured the fluorescence intensity
                            and OD600. Then the bacterial cultures were diluted with fresh M9 medium with antibiotics
                            and 10mM arabinose at a ratio of 1:100 once again and were grown for another 12h. Finally,
                            the fluorescence intensity and OD600 were measured once again.<br><br>
                            The ratio of fluorescence intensity/OD600 was calculated using the net fluorescence
                            intensity and net OD600. We regarded the ratio greater than 100 as “light” and the one less
                            than 100 as “dark”. The ratio of every well is shown below.<br><br>

                            <strong>A</strong>
                            <div align="center">
                                <div class="mdl-grid mdl-grid--no-spacing" style="margin: 0px">
                                    <div class="mdl-cell mdl-cell--6-col">
                                        <div class="demo-card-image mdl-card mdl-shadow--2dp"
                                             style="background: url('https://static.igem.org/mediawiki/parts/d/d0/2017data.png') center / cover; height: 240px; width: 400px">
                                            <div class="mdl-card__title mdl-card--expand"></div>
                                            <div class="mdl-card__actions">
                                                <span class="demo-card-image__filename">Animation in 96-well Plate</span>
                                            </div>
                                        </div>
                                    </div>
                                    <div class="mdl-cell mdl-cell--6-col">
                                        <div class="demo-card-image mdl-card mdl-shadow--2dp"
                                             style="background: url('https://static.igem.org/mediawiki/parts/2/2a/Peking_2017.png') center / cover; height: 240px; width: 400px">
                                            <div class="mdl-card__title mdl-card--expand"></div>
                                            <div class="mdl-card__actions">
                                                <span class="demo-card-image__filename"><font color="white">Animation in 96-well Plate</font></span>
                                            </div>
                                        </div>
                                    </div>
                                </div>
                            </div>
                            <br><br>
                            <strong>B</strong>
                            <div align="center">
                                <div class="mdl-grid mdl-grid--no-spacing" style="margin: 0px">
                                    <div class="mdl-cell mdl-cell--6-col">
                                        <div class="demo-card-image mdl-card mdl-shadow--2dp"
                                             style="background: url('https://static.igem.org/mediawiki/parts/3/3a/PKU_data.png') center / cover; height: 240px; width: 400px">
                                            <div class="mdl-card__title mdl-card--expand"></div>
                                            <div class="mdl-card__actions">
                                                <span class="demo-card-image__filename">Animation in 96-well Plate</span>
                                            </div>
                                        </div>
                                    </div>
                                    <div class="mdl-cell mdl-cell--6-col">
                                        <div class="demo-card-image mdl-card mdl-shadow--2dp"
                                             style="background: url('https://static.igem.org/mediawiki/parts/c/cf/Peking_PKU.png') center / cover; height: 240px; width: 400px">
                                            <div class="mdl-card__title mdl-card--expand"></div>
                                            <div class="mdl-card__actions">
                                                <span class="demo-card-image__filename"><font color="white">Animation in 96-well Plate</font></span>
                                            </div>
                                        </div>
                                    </div>
                                </div>
                            </div>
                            <br>

                            <strong>Figure 6.The pattern which transformed “2017” to “PKU”.</strong>A. When the culture
                            had begun for 12h, we got the pattern “2017”. B. When the culture had begun for 24h, we got
                            the pattern “PKU”.<br><br>


                            We have confirmed that our genetic circuits can work as designed through the “ 2017-PKU”
                            pattern transition. Next we constructed the system named “7 segment display”. The details of
                            the circuits in segments are shown in the table below.
                            <div align="center">
                                <img src="https://static.igem.org/mediawiki/2017/8/88/Peking_demo_1.jpeg" height="200px"/>
                                <img src="https://static.igem.org/mediawiki/2017/6/61/Peking_demo_2.jpeg" height="200px"/>
                                <img src="https://static.igem.org/mediawiki/2017/e/eb/Peking_demo_3.jpeg" height="200px"/><br>
                                <strong>Figure 7.The design of "1-2-3" transition</strong><br>
                            </div>


                            <br>

                            We got the figure below on cloud nine. The numbers in figure were fluorescence intensity of
                            corresponding wells, which were measured using the microplate reader mentioned above and
                            adjusted as the same methods. We defined the wells as “on” when its fluorescence intensity
                            exceeds 50; otherwise defined as “off”. As plain as the nose on your face, we managed to
                            make it. We realized the sequential logic!
                            <div align="center"> <img src="https://static.igem.org/mediawiki/2017/e/ee/Peking_123.png" height="350px"/>  <br><strong>Figure 8.The experiment result of "1-2-3" transition</strong><br></div>

                            <br>


                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">


                        <h2 id="pc6">References</h2>
                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Dyakonov T, Muir A, Nasri H, et al. Characterization of 582 natural and synthetic
                                terminators and quantification of their design constraints[J]. Nature Methods, 2013,
                                10(7):659-664.
                            </li>
                            <li>Brophy J A, Voigt C A. Antisense transcription as a tool to tune gene expression[J].
                                Molecular Systems Biology, 2016, 12(1):854.
                            </li>
                            <li>Yang L, Voigt CA, et al. Permanent genetic memory with >1-byte capacity. Nat Methods.
                                2014 Dec; 11(12):1261-6.
                            </li>
                            <li>Stothard P. The sequence manipulation suite: JavaScript programs for analyzing and
                                formatting protein and DNA sequences. Biotechniques. 2000 Jun; 28(6):1102, 1104.
                            </li>
                            <li>Lou C, Stanton B, Chen YJ, Munsky B, Voigt CA. Ribozyme-based insulator parts buffer
                                synthetic circuits from genetic context. Nat Biotechnol. 2012 Nov; 30(11):1137-42.
                                [PMID:23034349]
                            </li>
                        </ol>

                        <br><br><br><br><a
                            class="mdl-button mdl-button--accent mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                            href="#top" style="color: white;">
                        Back to top
                    </a>

                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->

                </div>

            </section>
            <section id="Clock" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">

                    <div class="mdl-card__supporting-text">

                        <h1>Clock</h1>
                        <br>


                        <section class="docs-toc docs-text-styling" style="margin-left: 0px">
                            <nav class="section-content" style="margin-left: 0px">
                                <ul style="margin-left: 0px">

                                    <li><a href="#pr1">Background</a></li>

                                    <li><a href="#pr2">Aims</a></li>
                                    <li><a href="#pr3">Key Achievements</a></li>
                                    <li><a href="#pr4">Our Approach</a></li>

                                    <li><a href="#pr5">Results</a></li>

                                    <li><a href="#pr6">References</a></li>

                                </ul>
                            </nav>
                        </section>

                        <h2 id="pr1">Background</h2>

                        <h3 id="pr1.1">Clock Signal</h3>
                        A clock signal is a particular type of signal that oscillates between a high and a low state
                        repeatedly. This signal is emitted by a clock generator and is utilized like a metronome to
                        trigger actions of a sequential logic circuit. In one clock signal cycle, the state of the
                        circuit, which is the basis of sequential logic circuits, remains unchanged after shifting. A
                        sequential logic circuit designed with an inner clock can change its state automatically, even
                        without inputs. Additionally, the time a state lasts can be controlled by the clock signal period.<br>
                        <br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/7/71/Peking_clock_figure1.png" height="300px"/></div>
                        <br>
                        <strong>Figure 1. Typical clock signal and fluorescence signals of the repressilator.</strong><br>
                        <br>

                        <h3 id="pr1.2">Repressilator Introduction</h3>
                        The repressilator is a synthetic genetic regulatory network consisting of a ring-oscillator
                        with three genes -- tetR from the Tn10 transposon,
                        <i>cI</i> from bacteriophage &lambda; and <i>lacI</i> from the <i>E. coli</i>
                        lactose operon, each expressing a protein that represses the next gene in the loop.<br>
                        <br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e7/Peking_clock_figure2.svg"
                                                 height="100px"/></div>
                        <br>
                        <strong>Figure 2. The structure of the original repressilator.</strong> It is encoded by a
                        low-copy plasmid with three repressor genes and a high-copy plasmid containing a GFP gene
                        driven by P<sub>LtetO1</sub>.<br>
                        <br>

                        <h3 id="pr1.3">The Repressilator as an Internal Clock</h3>
                        In our project, we aimed to use the repressilator to generate a clock signal. We chose it for
                        the following reasons:<br>
                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>It can generate up to three oscillatory signals in different phases; two different
                                signal peaks can drive the flip-flop to shift its state serving as a component of a period.</li>
                            <li>The period and amplitude of the oscillation can stay stable and robust through many
                                generations.</li>
                            <li>The period of the oscillation is adjustable. We can modify the oscillation period by
                                simply chang-ing the bacterial generation period and oscillator components.</li>
                            <li>It has the potential to be connected to a downstream circuit. Because of the oscillatory
                                changes in promoter strength, we can add the promoters (P<sub>R</sub> ,P<sub>LtetO1</sub>
                                ,P<sub>LlacO1</sub>) to the downstream circuit.</li>
                        </ol>
                        <br><br>

                    </div>

                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="pr2">Aims</h2>
                        Compared with natural biological systems, synthetic circuits such as the repressilator have a
                        lower accuracy of oscillation. There have been many efforts to enhance the robustness of
                        oscillation of the original repressilator, such as improvements in the core cellular processes
                        and the reporter systems . Thus, if we want to use the repressilator as an internal clock, we
                        have to determine whether our construct would have a significant influence on the oscillation
                        itself. To answer this question, we have to do the following experiments:<br>
                        <div class="nonumberitem">
                            <ul style="line-height: 2em;">
                                <li>Evaluate the repressilator system in our own experimental environment.</li>
                                <li>Construct an oscillator-induced protein expression system and compare its oscillation
                                    period with the parent repressilator.</li>
                            </ul>
                        </div>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="pr3">Key Achievements</h2>
                        <div class="nonumberitem">
                            <ul style="line-height: 2em;">
                                <li>Test the parent repressilator published in the 2016 paper by Potvin-Trottier <i>et al</i>.</li>
                                <li>Construct a recombinase oscillator and T7 oscillator.</li>
                            </ul>
                        </div>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="pr4">Our Approach</h2>

                        <h3 id="pr4.1">Reproduction of the Oscillation of the Original Repressilator</h3>
                        We used a stereoscope, a microfluidic device and a plate reader to reproduce the oscillation
                        experi-ments of the parent repressilator (single reporter oscillator and three-reporter
                        oscillator). The main strains we used in the oscillation-related experiments are listed in
                        Table 1. The clpXP genes in these strains were deleted to obtain more robust oscillations.<br>
                        <br>
                        <strong>Table1. The strains used in the oscillation-related experiments.</strong><br>
                        <div align="center">
                            <table class="mdl-data-table mdl-js-data-table">
                                <thead>
                                <tr>
                                    <th class="mdl-data-table__cell--non-numeric">Strain</th>
                                    <th>Plasmid</th>
                                    <th>Genotype (MC4100)</th>
                                </tr>
                                </thead>
                                <tbody>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Recombinase Oscillator</td>
                                    <td>plpt20-gp35<br>
                                        plpr41-reporter
                                    </td>
                                    <td>&Delta;<i>clp</i>XP</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Recombinase Amplifier</td>
                                    <td>plpt20-t7<br>
                                        plpt41-gp35-reporter
                                    </td>
                                    <td>&Delta;<i>clp</i>XP</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">T7 Polymerase Oscillator</td>
                                    <td>plpt20-t7<br>
                                        plpt41-t7 test
                                    </td>
                                    <td>&Delta;<i>clp</i>XP</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Triple Reporter Oscillator (LPT117)</td>
                                    <td>plpt107<br>
                                        plpt43
                                    </td>
                                    <td>&Delta;<i>clp</i>XP</td>
                                </tr>
                                <tr>
                                    <td class="mdl-data-table__cell--non-numeric">Single Reporter Oscillator</td>
                                    <td>plpt20<br>
                                        Plpt41
                                    </td>
                                    <td>&Delta;<i>clp</i>XP</td>
                                </tr>
                                </tbody>
                            </table>
                        </div>
                        <br>

                        Since one of the repressors is under the control of P<sub>LlacO1</sub>, we can use IPTG
                        (Isopropyl &beta;-D-Thiogalactoside) to synchronize the oscillation in different cells. We also
                        compared the synchronized oscillation and the unsynchronized oscillation. (see Results figure)<br>
                        <br>
                        If we want to look at dynamic changes of the oscillation, population-based experiments are not
                        enough, so we also designed a microfluidic device of the same type as the "mother machine"
                        used in our lab to house single cells and their offspring. In this device, cells are trapped
                        in narrow channels and newborn cells are washed away by fresh medium. (see Results video1)<br>

                        <br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/b/b6/Peking_clock_fig3.png" height="400px"/>
                        </div>
                        <strong>Figure 3. Design of the "Mother Machine".</strong> It consists of dozens of thin channels
                        holding single cells, and can be used to track individual cell line for generations.<br>
                        <br><br>

                        <h3 id="pr4.2">Design of the Recombinase Oscillator</h3>
                        In order to test whether our modifications of the repressilator would severely affect its
                        oscillation pattern, we combined the repressilator with recombinases (Bxb1 gp35) to obtain an
                        oscillator-activated recombinase expression system. By adjusting this system, we can also
                        determine whether the oscillation period is compatible with the recombination time.<br>
                        <br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e7/Peking_clock_fig4_recombinaseschematic.svg" height="600px"/>
                        </div><br>

                        <strong>Figure 4. Schematic diagram of the recombinase oscillator.</strong><br>
                        <br>
                        For the expression system of the recombinase oscillator, we made use of the plpt20 plasmid
                        (Laurent <i>et.al.</i>, 2016), and we cloned Bxb1 gp35 downstream of mVenus in plpt20. By doing
                        this, the expression of recombinase was coupled to the expression of mVenus, which is under the
                        control of the promoter P<sub>LtetO1</sub>.<br>
                        As for the reporter system used to detect recombinase expression, we constructed a revertible
                        promoter surrounded by a Bxb1 attB site and a Bxb1 attP site on the sponge plasmid. When the
                        recombinase is expressed, it inverts the promoter sequence and mRFP is expressed.<br>
                        Both the expression and reporter systems of the recombinase oscillator were constructed
                        using Gibson assembly.<br>
                        <br>
                        <strong>A</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/3/32/Peking_clock_fig5a_recombinase.svg" height="100px"/></div>
                        <br>
                        <strong>B</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/d/d1/Peking_clock_figure5b.svg" height="200px"/></div>
                        <br>

                        <strong>Figure 5. Plasmids of the recombinase oscillator.</strong>  (A) Plasmid encoding the oscillatory recombinase expression system (plpt20-gp35).
                        (B) The plasmid encoding the oscillatory recombinase reporter system (plpt41-reporter).<br>
                        <br>

                        <h4>Possible improvements</h4>

                        <div class="nonumberitem">
                            <ul style="line-height: 2em;">
                                <li>mRFP is a widely used fluorescent protein in mammalian cells, but it has its
                                    drawbacks. As we have discovered in our experiments, mRFP takes a rather long time
                                    to mature and emit red fluorescence. Thus, we may have to exchange the fluorescent
                                    reporter protein for a fast-maturing one such as mKate.</li>
                                <li>The oscillation period is not compatible with the duration of recombinase-induced
                                    promoter reversal and mRFP maturation. In order to adjust the oscillation period,
                                    we can use different liquid media to prolong the oscillation period. Previous work
                                    has shown that the oscillation period is proportional to the cell division time. Thus,
                                    by using a liquid medium such as M9, we can increase the cell division time and
                                    prolong the oscillation period.</li>
                            </ul>
                        </div>
                        <br>

                        <h3 id="pr4.3">Design of the T7-coupled Oscillator</h3>
                        The repressilator is constantly working as an internal clock, but under most circumstances
                        we would like to start the internal clock in a specific moment. Using IPTG to synchronize
                        the internal clocks of all the cells in a population and keep them in the same phase seems
                        plausible. However, through our experiments with the recombinase oscillator we found that
                        using IPTG cannot prevent signal leakage of the internal clock. Thus, we would like to add an
                        "AND" gate behind the clock signal, so that we can turn on or turn off the internal clock.<br>

                        T7 RNA polymerase has been previously used to implement an "AND" gate. In that design, the
                        first promoter controls the expression of a T7 RNA polymerase gene with two internal amber
                        stop codons. The second promoter controls the amber suppressor tRNA supD. When both promoters
                        are activated, T7 RNA polymerase is expressed and can act specifically on the downstream T7
                        promoter. What's more, T7 RNA polymerase can be used as an amplifier to increase the signal
                        amplitude.<br>
                        <br>
                        Our aim is to prove that T7 RNA polymerase can be combined with the repressilator and can serve
                        as a potential on-off switch and amplifier. Therefore, we designed a T7 oscillator and
                        recombinase amplifier.<br>
                        <br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/f/f8/Peking_clock_fig6_t7schematic.svg" height="600px"/></div>
                        <br>
                        <strong>Figure6. Schematic diagram of the recombinase oscillator.</strong><br>
                        <br>

                        <strong>A</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/8/8b/Peking_clock_fig7a_t7.svg" height="100px"/></div>
                        <br>

                        <strong>B</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/0/04/Peking_clock_figure7b.svg" height="200px"/></div>
                        <br>

                        <strong>C</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/6/65/Peking_clock_figure7c.svg" height="150px"/></div>
                        <br>


                        <strong>Figure 7. Plasmids of the T7 polymerase oscillator.</strong> (A) The plasmid encoding the T7 oscillator expression system (plpt20-T7). (B) The
                        plasmid encoding the T7 oscillator reporter system (plpt41-T7 test). (C) The plasmid encoding
                        the recombinase amplifier system.<br>
                        <br>
                        For the expression system of the T7 oscillator, we also used the plpt20 plasmid (Laurent <i>et.al.</i>
                        , 2016). We cloned the T7 RNA polymerase right behind mVenus in plpt20. To detect the
                        expression of T7 RNA polymerase, we added a T7 promoter before mRFP in the plpt41 plasmid.
                        In order to prove that T7 polymerase can be used as an amplifier, we also constructed a
                        recombinase amplifier. The re-porter system of the recombinase amplifier was adapted from the
                        plpt41-reporter; we added a Bxb1-gp35 activated by a T7 promoter in the plpt41-reporter
                        plasmid.<br>
                        <br>

                        <h3 id="pr4.4">Microfluidic Image Analysis</h3>
                        Firstly, we opened the files from the microscope generated in nd2 format in NIS element viewer
                        or image J plugins NDi6d. We were then able to change them to videos or pictures in Tag Image
                        File Format. Finally, we used image J to analyze the intensity of the images in TIF format.<br>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="pr5">Results</h2>

                        <h3 id="pr5.1">Recurrence of the Oscillation in the Repressilator</h3>

                        <div class="mdl-grid mdl-grid--no-spacing" style="margin: 0px">
                            <div class="mdl-cell mdl-cell--6-col">
                                <div class="demo-card-image mdl-card mdl-shadow--2dp" style="background: url('https://static.igem.org/mediawiki/2017/5/5b/Peking_clock_figure8_1.png') center / cover; height: 500px; width: 400px">
                                    <div class="mdl-card__title mdl-card--expand"></div>
                                    <div class="mdl-card__actions">
                                        <span class="demo-card-image__filename"></span>
                                    </div>
                                </div>
                            </div>
                            <div class="mdl-cell mdl-cell--6-col">
                                <div class="demo-card-image mdl-card mdl-shadow--2dp" style="background: url('https://static.igem.org/mediawiki/2017/1/15/Peking_clock_figure8_2.png') center / cover; height: 500px; width: 400px">
                                    <div class="mdl-card__title mdl-card--expand"></div>
                                    <div class="mdl-card__actions">
                                        <span class="demo-card-image__filename"></span>
                                    </div>
                                </div>
                            </div>
                        </div><br>

                        <strong>Figure 8. Stereoscopic image of colony oscillation.</strong> Triple reporter Plpt107+titration sponge.
                        We repeated the experiments of the original repressilator (John et al., 2016,) using the LPT117
                        strain. By using a stereoscope, we discovered concentric yellow fluorescence circles in the
                        colony of LPT117. This spatial pattern is the reflection of temporal pattern of the oscillation.
                        It is formed when the fluorescence signal oscillates during cell division, and inner parts of
                        cells in the colony cannot divide and remain the fluorescence signal.<br>
                        We repeated the experiments of the original repressilator (John <i>et al.</i>, 2016,) using the LPT117
                        strain. By using a stereoscope, we discovered concentric green fluorescence circles in the
                        colony of LPT117.<br>
                        <br>

                        <strong>A</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/ea/Peking_clock_figure9a_1.png" height="200px"/></div>
                        <br>

                        <strong>B</strong><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/1/1a/Peking_clock_figure9b.png" height="200px"/></div>
                        <br>


                        <strong>Figure 9. Typical time trace of a single cell for original repressilator.</strong> A. Time trace of LPT117
                        repressilator (plpt107+titration sponge). B. Time trace of single reporter repressilator
                        (plpt20+titration sponge). Both traces were normalized to their means. The oscillation period of
                        LPT117 is around 510 minutes in LB medium,14 generations ( cell division period is about 35 minutes). The oscillation period of the single reporter oscillator
                        is also around 15 generations, 450 minutes (cell division period is about 30 minutes).<br>

                        <br>
                        <div align="center">
                            <video width="602px" height="345px" controls="controls">

                                <source src="https://static.igem.org/mediawiki/2017/8/89/Peking_video1_LPT117.webm" type="video/webm"></source>
                                <source src="https://static.igem.org/mediawiki/2017/8/89/Peking_video1_LPT117.webm" type="video/ogg"></source>
                            </video>
                        </div>
                        <br>

                        <div align="center">
                            <strong>Video 1. Microfluidic videos of LPT117 oscillations in the mother machine.</strong> <br><br>
                        </div>

                        <h3 id="pr5.2">Recombinase Oscillator</h3>

                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/4/4b/Peking_clock_recombinasevideo.mp4 "type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/4/4b/Peking_clock_recombinasevideo.mp4" type="video/ogg"></source>

                            </video>
                        </div><br>

                        <div align="center">
                            <strong>Video 2. Dynamic changes of a bacteria colony of recombinase oscillator.</strong> At the beginning, the colony was green because of the expression of GFP.When the colony had grown to a certain size, Bxb1-gp35 was expressed leading the expression of mRFP..
                            <br><br>
                        </div>

                        <br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/1/1b/Peking_clock_fig10.png" height="300px"/></div>
                        <br>

                        <strong>Figure 10. Optical microscopic views of a bacteria colony with a ' disk-like ' pattern.</strong> An optical
                        microscopic view of a bacterial colony with a  ' disk-like ' pattern, the red parts of the colony are distributed in wedge
                        form. When recombinase Bxb1-gp35 expresses, the promoter of mRFP surrounded by Bxb1 attB and attP sites is inverted, which leads to the
                        expression of mRFP.Some cells shift from yellow to red because of the expression of Bxb1-gp35, and its offsping, which are distributed in wedge form, kept expressing mRFP..<br>
                        <br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/2/27/Peking_clock_figure10b.png"
                                                 height="450px"/></div>
                        <br>

                        <strong>Figure 11. Time trace of a single cell for recombinase oscillator.</strong> The oscillation period of
                        recombinase oscillator is around 300 minutes, 14 gene in LB medium (cell division period is 20 minutes), which is similar to the oscillation
                        period of LPT117 and single reporter repressilator. These results shows that our modification
                        on the parent repressilator (combine recombinase with repressilator) will not affect the clock
                        itself and clock signal can be passed from the repressilator to downstream structure (invert of
                        mRFP promoter).<br>
                        <br>

                        <div align="center">
                            <video width="602px" height="345px" controls="controls">
                                <source src="https://static.igem.org/mediawiki/2017/f/f1/Peking_video2_plpt20_GP35.webm "type="video/mp4"></source>
                                <source src="https://static.igem.org/mediawiki/2017/f/f1/Peking_video2_plpt20_GP35.webm" type="video/ogg"></source>

                            </video>
                        </div><br>
                        <strong>Video 3. Microfluidic video of Recombinase Oscillator.</strong><br>
                        <br>


                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">



                        <h2 id="pr6">References</h2>
                        <ol start="1">
                            <script type="text/javascript">
                                (function () {

                                    var doc_ols = document.getElementsByTagName("ol");

                                    for (i = 0; i < doc_ols.length; i++) {

                                        var ol_start = doc_ols[i].getAttribute("start") - 1;
                                        doc_ols[i].setAttribute("style", "counter-reset:ol " + ol_start + ";");

                                    }

                                })();
                            </script>

                            <li>Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional
                                regulators. Na-ture, 403(6767), 335-338.</li>
                            <li>Potvin-Trottier, L., Lord, N. D., Vinnicombe, G., & Paulsson, J. (2016). Synchronous
                                long-term oscilla-tions in a synthetic gene circuit. Nature, 538(7626), 514-517.</li>
                            <li>Wang, P., Robert, L., Pelletier, J., Dang, W., Taddei, F., Wright, A., & Jun, S. (2010).
                                Robust Growth of Escherichia coli. Current Biology, 20(12), 1099–1103.</li>
                        </ol>

                        <br><br><br><br><a class="mdl-button mdl-button--accent mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect" href = "#top" style="color: white;">
                        Back to top
                    </a>



                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->

                </div>

            </section>
            <section id="Demonstrate" class="mdl-components__page mdl-grid">
                <div class="demo-card-wide mdl-card mdl-shadow--2dp">

                    <div class="mdl-card__supporting-text">

                        <h1>Demostrate</h1>
                        <br>


                        <section class="docs-toc docs-text-styling" style="margin-left: 0px">
                            <nav class="section-content" style="margin-left: 0px">
                                <ul style="margin-left: 0px">

                                    <li><a href="#de1">Background & Design</a></li>
                                    <li><a href="#de2">Methods</a></li>
                                    <li><a href="#de3">Results</a></li>
                                    <li><a href="#de4">Discussion</a></li>
                                    <li><a href="#de5">Potential Applications</a></li>


                                </ul>
                            </nav>
                        </section>

                        <h2 id="de1">Background & Design</h2>
                        We attempts to develop a framework of biological sequential circuits that are programmable. The
                        envisioned
                        circuit is capable of both storing states in DNA and automatically running a series of
                        instructions in a
                        specific order. More specifically, the sequential logic (or a real-state machine) that consists
                        of a clock
                        (trigger signal), flip flop (remembering device) and control unit (functional part) in bacteria.
                        The clock
                        is an oscillator with a repeated signal cycle that is utilized like a metronome to trigger
                        actions of
                        sequential logic circuits. Flip-flop is a memory device that can remember a state. Paired with a
                        clock
                        signal, it can realize state transition. The control unit is a functional part which can convert
                        a signal
                        from flip-flop into complex functions. With such a design, historical events are recorded and
                        influence
                        current cell behavior.<br>
                        <br><br>

                    </div>

                    <div class="mdl-card__menu">
                        <button class="mdl-button mdl-button--icon mdl-js-button mdl-js-ripple-effect">
                            <i class="material-icons">star</i>
                        </button>
                    </div>

                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="de2">Methods</h2>
                        The bio-flip-flop is constructed as two sequences each with an invertible promoter flanked
                        between
                        recombination sites and expressing recombinase or recombinase-RDF fusion protein mediating
                        inversion of
                        another sequence.<br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e6/Peking_demostrate_fig1.png"
                                                 height="100px"/></div>
                        <br>
                        <strong>Fig.1. Bio-flip-flop construction.</strong><br>
                        <br>

                        We first tested and optimized recombinase and recombinase-RDF fusion protein functions and we
                        standardized
                        these parts into BioBrick devices. Then we separated the function of bio-flip-flops and built
                        two circuits:
                        the Forward and Backward latch. Each of them can implement state change when receives a group of
                        trigger
                        signal consisting of two inducing events. We then introduced these latches in vivo and proved
                        their
                        functionality.<br>
                        Check the <a href="https://2017.igem.org/Team:Peking/Project#Flip-flop" target="blank"
                                     style="color: #226CE4"><strong>FLIP-FLOP</strong></a>
                        module for more details. <br><br>

                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/e/e3/Peking_flipflop_fig_10.svg"
                                                 width="600px"/></div>
                        <br><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/7/7b/Peking_flipflop_fig_12.svg"
                                                 width="600px"/></div>
                        <br>
                        <strong>Fig.2. Construction of Forward and Backward latches of the bio-flip-flop and their
                            function.</strong><br>
                        <br>

                        Unidirectional terminators whose directions are changed by recombinases or recombinase-RDF
                        fusion proteins
                        are used to control transcription of the target gene. We measured the strengths of 24
                        terminators in both
                        directions, and we elected six suitable unidirectional terminators. We measured the strengths of
                        several
                        terminators paired with attB/P sites and the inversion efficiency of three recombinases.<br>
                        Check the <a href="https://2017.igem.org/Team:Peking/Project#Controller" target="blank"
                                     style="color: #226CE4"><strong>CONTROLLER</strong></a>
                        module for more details. <br><br>
                        <div align="center"><img src="https://static.igem.org/mediawiki/2017/a/a2/2nd_generation_Ts.png"
                                                 height="500px"/></div>
                        <br><br>

                        We used repressilator, a classic genetic oscillator, as an internal clock to drive our
                        bio-flip-flop. To do
                        so, we first observed and evaluated the repressilator system in our own experimental
                        environment. We then
                        constructed an oscillator-induced recombinase expression system and observed promoter inversion,
                        which shows
                        that repressilator has the possibility to act as an internal oscillatory trigger signal
                        generator.<br>
                        Check the <a href="https://2017.igem.org/Team:Peking/Project#Clock"
                                     style="color: #226CE4"><strong>CLOCK</strong></a>
                        module for more details.

                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="de3">Results</h2>

                        Following the methodology of modularity in synthetic biology, we divided the system into three
                        key
                        components. We showed the feasibility of their assembly into a whole system by a set of
                        simulation
                        results.<br>
                        <br>
                        First, an ordinary differential equation system describing recombinase and RDF function was
                        built.
                        Parameters were estimated from experimental data. Then we assembled four components (two
                        recombinases and
                        two fusion proteins) as the one in flip-flop design. Inducing of the system is in the form of
                        square waves
                        to simulate manual induction like changing of medium.<br>
                        <br>


                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/8/87/Peking_demostrate_fig3_1.png"
                                height="300px"/></div>
                        <br><br>
                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/7/73/Peking_demostrate_fig3_2.png"
                                height="150px"/></div>
                        <br><br>

                        <strong>Fig.3. Simulation of the whole flip-flop function.</strong> X_F denotes the fraction of
                        forward direction (right) of
                        the upper promoter pBAD. Y_F denotes the fraction of forward direction (right) of the lower
                        promoter pTAC.<br>
                        <br>
                        We also proved that the system can also be triggered by repressilator as internal clock signal.
                        Substituting
                        the upper promoter (repressed by tetR protein) to PLtetO1 and the lower one (repressed by cI
                        protein) to PR,
                        we then did another simulation of forward latch and proved that the flip-flop can change its
                        state and then
                        recover back to initial state after arrival of two adjacent trigger signal groups.<br>

                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/e/ef/Peking_demostrate_fig4_1.png"
                                height="400px"/></div>
                        <br><br>
                        <div align="center"><img
                                src="https://static.igem.org/mediawiki/2017/b/ba/Peking_demostrate_fig4_2.png"
                                height="400px"/></div>
                        <br><br>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="de4">Discussion</h2>
                        In this simulation experiment, the flip-flop lost approximately 10% of its initial state after
                        two trigger
                        signal groups. If we use manual and discrete induction, the loss is roughly 5%. These results
                        have reached
                        the expectations. However, in practical experiment, there are still many problems to solve and
                        further
                        research is needed. Though we constructed and configured a set of recombinase and
                        recombinase-RDF fusion
                        protein tools, we observed leaky expression and other problems when assembled. Further
                        modification of
                        repressilator (adding one more recombinase) has to be tested to satisfy the need of in vivo
                        automatic state
                        transition.<br>
                        <br><br>

                    </div>
                </div>

                <div class="demo-card-wide mdl-card mdl-shadow--2dp">


                    <div class="mdl-card__supporting-text">

                        <h2 id="de5">Potential Applications</h2>
                        This work tries to point the way toward building large computational systems from modular
                        biological
                        parts—basic sequential computing devices in living cells—and ultimately, programming complex
                        biological
                        functions. We also illustrated the possibility of self-triggered state transition. Possible
                        application
                        would be an automatic cell factory that can complete a series of manufacturing procedures with
                        just one
                        trigger at the start. Furthermore, our bio-flip-flop can act as an in vivo memory device and aid
                        in
                        recording cellular events, which can provide deep insight into cell differentiation and
                        development.<br>

                        <br><br><br><br><a
                            class="mdl-button mdl-button--accent mdl-js-button mdl-button--raised mdl-button--accent mdl-js-ripple-effect"
                            href="#top" style="color: white;">
                        Back to top
                    </a>

                    </div>
                    <!--<div class="mdl-card__actions mdl-card--border">
                        <a class="mdl-button mdl-button--colored mdl-js-button mdl-js-ripple-effect">
                            Get Started
                        </a>
                    </div>-->

                </div>

            </section>


        </div>
    </div>
    <section class="docs-text-styling download mdl-color--grey-800" style="height: 1px"></section>
    <footer class="mdl-mega-footer" style="height: 260px; margin-bottom: 0px;">
        <div class="mdl-mega-footer__middle-section">

            <div class="mdl-mega-footer__drop-down-section">
                <input class="mdl-mega-footer__heading-checkbox" type="checkbox" checked>
                <h1 class="mdl-mega-footer__heading">Acknowledgement</h1>
                <ul class="mdl-mega-footer__link-list" style="margin-left: 0px;>
                    <li><a href=" http:
                //dean.pku.edu.cn/pkudean/"> </li>
                <li><a href="http://dean.pku.edu.cn/pkudean/"><img
                        src="https://static.igem.org/mediawiki/2017/9/99/Peking_footer_logo_admin.png" height="50px"/></a>
                </li>
                <li><a href="http://www.bio.pku.edu.cn/en/"> <img
                        src="https://static.igem.org/mediawiki/2017/a/ab/Peking_footer_logo_slslogo.png" height="50px"/> </a>
                </li>
                <li><a href="http://cqb.pku.edu.cn/en/"> <img
                        src="https://static.igem.org/mediawiki/2017/9/9a/Peking_footer_logo_cqb.png" height="48px"/> </a></li>

                </ul>
            </div>

            <div class="mdl-mega-footer__drop-down-section">
                <input class="mdl-mega-footer__heading-checkbox" type="checkbox" checked>
                <h1 class="mdl-mega-footer__heading">Connection</h1>
                <ul class="mdl-mega-footer__link-list" style="margin-left: 0px;>
                    <li><a href=" https:
                //www.facebook.com/pekingigem"></a></li>
                <li><a href="https://www.facebook.com/pekingigem">Facebook</a></li>
                <li><a href="https://www.youtube.com/channel/UCefEYVCVqKJLmoJrSxivgZQ">Youtube</a></li>
                <li><a href="https://twitter.com/iGEM_Peking">Twitter</a></li>
                <li><a href="https://github.com/PekingiGEM2017">Github</a></li>
                </ul>
            </div>

            <div class="mdl-mega-footer__drop-down-section">
                <input class="mdl-mega-footer__heading-checkbox" type="checkbox" checked>
                <h1 class="mdl-mega-footer__heading">Contact</h1>
                <ul class="mdl-mega-footer__link-list" style="margin-left: 0px;>
                    <li><a href=" http:
                //www.synbiowiki.com"> </a></li>
                <li><a href="http://www.synbiowiki.com">SynBioWiki.com</a></li>
                <li><a href="mailto:PekingIGEM@gmail.com">Mail</a></li>
                </ul>
            </div>

            <div class="mdl-mega-footer__drop-down-section">
                <input class="mdl-mega-footer__heading-checkbox" type="checkbox" checked>
                <h2 style="color: white; "><strong>Peking iGEM</strong> 2017</h2>
                <ul class="mdl-mega-footer__link-list" style="margin-left: 0px;">
                    <li style="color: white; line-height: 1.88em;">Peking University,<br> No.5 Yiheyuan Road Haidian
                        District, Beijing, P.R.China<br>100871
                    </li>
                </ul>
            </div>

        </div>


    </footer>

</main>


</body>
</html>