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        <h1>Present Challenges of<br/> Experimenting With Plasmid Systems</h1>
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        <div class="presentation">
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            <div class="left">
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                <div class="left-steps">
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                    <div class="left-step is-current" style="display: block">
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                        <div class="step">1. 1.</div>
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                        <div class="title">Plasmid Replication </div>
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                        <p>Origin of replication (Ori) is a sequence where the plasmid replication begins. Plasmids with similar Ori sites use the same replication machinery. </p>
 +
                        <h6>Problem #1</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">1. 2.</div>
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                        <div class="title">Unbalanced Replication</div>
 +
                        <p> Replication machinery does not discriminate between similar Ori sites, therefore smaller plasmids are replicated faster and rapidly outgrow larger plasmids, which leads to uneven plasmid ratios in a cell.</p>
 +
                        <h6>Problem #1</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">1. 3.</div>
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                        <div class="title">Plasmid Loss</div>
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                        <p> This is even more likely to occur when some plasmids are low copy number. When the cell
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                            divides, low copy plasmids tend to be lost in next generations. </p>
 +
                        <h6>Problem #1</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">2. 1.</div>
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                        <div class="title">Plasmid Incompatibility </div>
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                        <p>This inability to stably co-maintain different plasmids over a number of generations in the same bacterial cell line is called plasmid incompatibility. One needs to use different types of Ori to avoid this trouble.</p>
 +
                        <h6>Problem #2</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">2. 2.</div>
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                        <div class="title">Plasmid Incompatibility</div>
 +
                        <p>However, it often causes trouble due to limited Ori selection options or the lack of good combination of Ori and plasmid's copy number. Imagine you require a system consisting of two - low and high copy number  plasmids. Try picking them from the list.</p>
 +
                        <h6>Problem #2</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">2. 3.</div>
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                        <div class="title">Fixed Copy Number</div>
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                        <p>Currently no handy method of changing the copy number of particular plasmid exists. It must be either low, or medium, or high.</p>
 +
                        <h6>Problem #2</h6>
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                    </div>
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                    <div class="left-step">
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                        <div class="step">2. 4.</div>
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                        <div class="title">Deficient Responsivity</div>
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                        <p>There is also no mechanism to adjust the copy number when so is required in any specific
 +
                            situation, for example: incoming cellular signals or special patterns.</p>
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                        <h6>Problem #2</h6>
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                    </div>
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                        <div class="content visible">
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                            <img id="p12" src="https://static.igem.org/mediawiki/2017/1/1d/T--Vilnius-Lithuania--step-1-2-center1.gif">
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                            <img src="https://static.igem.org/mediawiki/2017/5/52/T--Vilnius-Lithuania--step-2-1-image1.png" width="381" height="185">
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                            <div class="success-text" style="display: none">Success!</div>
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                            <div class="error-text" style="display: none">Error!</div>
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                            <div class="interactive-plasmid suc-err">
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                                <img src="https://static.igem.org/mediawiki/2017/4/42/T--Vilnius-Lithuania--step-2-2-dna.png" class="dna">
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                                <img src="https://static.igem.org/mediawiki/2017/5/58/T--Vilnius-Lithuania--step-2-2-int1.png" class="int1" width="42"
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                                <img src="https://static.igem.org/mediawiki/2017/f/f0/T--Vilnius-Lithuania--step-2-2-int2.png" class="int2" width="42"
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                                <img src="https://static.igem.org/mediawiki/2017/8/8a/T--Vilnius-Lithuania--step-2-2-int3.png" class="int3" width="42"
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                                <img src="https://static.igem.org/mediawiki/2017/3/35/T--Vilnius-Lithuania--step-2-2-int4.png" class="int4" width="42"
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                                <img src="https://static.igem.org/mediawiki/2017/4/44/T--Vilnius-Lithuania--step-2-2-int5.png" class="int5" width="42"
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                            </div>
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                            <div class="success-text-long" style="display: none">
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                                As you can see from our example, even when one knows what copy number is needed for system, it is hard to find the right combination of compatible replicons and right copy number.
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                            </div>
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                            <div class="error-text-long" style="display: none">
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                                <div>Plasmids are incompatible, because they use the same replicons.</div>
 +
                                <div class="button">Try again</div>
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                            </div>
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                        <div class="content">
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                            <img class="i23center active" src="https://static.igem.org/mediawiki/2017/7/72/T--Vilnius-Lithuania--step-2-3-centerhigh.png" width="381" height="185">
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                            <img class="i23center" src="https://static.igem.org/mediawiki/2017/3/3a/T--Vilnius-Lithuania--step-2-3-centermedium.png" width="381" height="185">
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                            <img class="i23center" src="https://static.igem.org/mediawiki/2017/6/61/T--Vilnius-Lithuania--step-2-3-centerlow.png" width="381" height="185">
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                        <div class="content">
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                            <div class="interactive-plasmid">
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                                <div class="wave">
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                                    <img src="https://static.igem.org/mediawiki/2017/2/2d/T--Vilnius-Lithuania--step-2-4-wave.png" width="94" height="63">
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                                </div>
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                                <img src="https://static.igem.org/mediawiki/2017/6/66/T--Vilnius-Lithuania--step-2-4-plasmid1.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/a/a6/T--Vilnius-Lithuania--step-2-4-plasmid2.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/3/39/T--Vilnius-Lithuania--step-2-4-plasmid3.png" class="plasmid" width="50" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/6/66/T--Vilnius-Lithuania--step-2-4-plasmid1.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/a/a6/T--Vilnius-Lithuania--step-2-4-plasmid2.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/3/39/T--Vilnius-Lithuania--step-2-4-plasmid3.png" class="plasmid" width="50" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/6/66/T--Vilnius-Lithuania--step-2-4-plasmid1.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/a/a6/T--Vilnius-Lithuania--step-2-4-plasmid2.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/3/39/T--Vilnius-Lithuania--step-2-4-plasmid3.png" class="plasmid" width="50" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/6/66/T--Vilnius-Lithuania--step-2-4-plasmid1.png" class="plasmid" width="48" height="48">
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                                <img src="https://static.igem.org/mediawiki/2017/4/42/T--Vilnius-Lithuania--step-2-2-dna.png" class="dna">
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                            </div>
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                        <div class="content">
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                            <h2>Did this look smart, easy or convenient?</h2>
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                            <p>We think it is time for a change.</p>
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                        </div>
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                    </div>
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                </div>
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                <div class="mob-prev" style="background-image: url('https://static.igem.org/mediawiki/2017/a/a0/T--Vilnius-Lithuania--step-1-1-arrow.png')"></div>
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                <div class="mob-next" style="background-image: url('https://static.igem.org/mediawiki/2017/a/a0/T--Vilnius-Lithuania--step-1-1-arrow.png')"></div>
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            </div>
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            <div class="right">
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                <div class="right-steps">
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                    <div class="right-step is-current" style="display: block">
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                        <div class="box">
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/f/fa/T--Vilnius-Lithuania--step-1-1-boxline1.png" width="42"
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                                </div>
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                                <div class="box-line-name">Plasmid A</div>
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                            </div>
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/1/1a/T--Vilnius-Lithuania--step-1-1-boxline2.png" width="42"
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                                        height="42">
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                                </div>
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                                <div class="box-line-name">Plasmid B</div>
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                            </div>
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/f/f9/T--Vilnius-Lithuania--step-1-1-boxline3.png" width="12"
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                                        height="12">
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                                </div>
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                                <div class="box-line-name">Origin of replication</div>
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                            </div>
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/T--Vilnius-Lithuania--step-1-1-boxline4.png" width="19"
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                                        height="19">
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                                </div>
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                                <div class="box-line-name">Replication machinery</div>
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                            </div>
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                        </div>
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                    </div>
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                    <div class="right-step">
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                        <div class="box">
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/f/fa/T--Vilnius-Lithuania--step-1-1-boxline1.png" width="42"
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                                        height="42">
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                                </div>
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                                <div class="box-line-name">Plasmid A</div>
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                            </div>
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                            <div class="box-line">
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                                <div class="imgcont">
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                                    <img src="https://static.igem.org/mediawiki/2017/1/1a/T--Vilnius-Lithuania--step-1-1-boxline2.png" width="42"
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                                        height="42">
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                                </div>
 +
                                <div class="box-line-name">Plasmid B</div>
 +
                            </div>
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/f9/T--Vilnius-Lithuania--step-1-1-boxline3.png" width="12"
 +
                                        height="12">
 +
                                </div>
 +
                                <div class="box-line-name">Origin of replication</div>
 +
                            </div>
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/T--Vilnius-Lithuania--step-1-1-boxline4.png" width="19"
 +
                                        height="19">
 +
                                </div>
 +
                                <div class="box-line-name">Replication machinery</div>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="right-step">
 +
                        <div class="box">
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/fa/T--Vilnius-Lithuania--step-1-1-boxline1.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="box-line-name">Plasmid A</div>
 +
                            </div>
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/1/1a/T--Vilnius-Lithuania--step-1-1-boxline2.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="box-line-name">Plasmid B</div>
 +
                            </div>
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/f9/T--Vilnius-Lithuania--step-1-1-boxline3.png" width="12"
 +
                                        height="12">
 +
                                </div>
 +
                                <div class="box-line-name">Origin of replication</div>
 +
                            </div>
 +
                            <div class="box-line">
 +
                                <div class="imgcont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/T--Vilnius-Lithuania--step-1-1-boxline4.png" width="19"
 +
                                        height="19">
 +
                                </div>
 +
                                <div class="box-line-name">Replication machinery</div>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="right-step">
 +
                        <div class="box">
 +
                            <div class="box-title">Different plasmids</div>
 +
                            <div class="box-plasmids">
 +
                                <div class="box-plasmid">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/fa/T--Vilnius-Lithuania--step-1-1-boxline1.png" width="42"
 +
                                        height="42">
 +
                                    <p>A</p>
 +
                                </div>
 +
                                <div class="box-plasmid">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/39/T--Vilnius-Lithuania--step-2-1-plasmid1.png" width="42"
 +
                                        height="42">
 +
                                    <p>B</p>
 +
                                </div>
 +
                                <div class="box-plasmid">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/b/b4/T--Vilnius-Lithuania--step-2-1-plasmid2.png" width="42"
 +
                                        height="42">
 +
                                    <p>C</p>
 +
                                </div>
 +
                                <div class="box-plasmid">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Vilnius-Lithuania--step-1-2-boxline2.png" width="42"
 +
                                        height="42">
 +
                                    <p>D</p>
 +
                                </div>
 +
                            </div>
 +
                        </div>
 +
                        <div class="box">
 +
                            <div class="box-title">Different types of ORI</div>
 +
                            <div class="box-sb">
 +
                                <img src="https://static.igem.org/mediawiki/2017/7/78/T--Vilnius-Lithuania--step-2-1-ori1.png" width="12" height="12">
 +
                                <img src="https://static.igem.org/mediawiki/2017/9/9e/T--Vilnius-Lithuania--step-2-1-ori2.png" width="14" height="12">
 +
                                <img src="https://static.igem.org/mediawiki/2017/5/51/T--Vilnius-Lithuania--step-2-1-ori3.png" width="12" height="12">
 +
                                <img src="https://static.igem.org/mediawiki/2017/a/a9/T--Vilnius-Lithuania--step-2-1-ori4.png" width="15" height="12">
 +
                            </div>
 +
                        </div>
 +
                        <div class="box">
 +
                            <div class="box-title">Different replication machinery molecules</div>
 +
                            <div class="box-sb">
 +
                                <img src="https://static.igem.org/mediawiki/2017/7/74/T--Vilnius-Lithuania--step-2-1-mol1.png" width="18" height="18">
 +
                                <img src="https://static.igem.org/mediawiki/2017/2/23/T--Vilnius-Lithuania--step-2-1-mol2.png" width="21" height="18">
 +
                                <img src="https://static.igem.org/mediawiki/2017/5/5d/T--Vilnius-Lithuania--step-2-1-mol3.png" width="18" height="18">
 +
                                <img src="https://static.igem.org/mediawiki/2017/7/7b/T--Vilnius-Lithuania--step-2-1-mol4.png" width="21" height="18">
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="right-step">
 +
                        <div class="interaction22">
 +
                            <div class="i22-heading">
 +
                                <p>Plasmid</p>
 +
                                <p>Copy number</p>
 +
                            </div>
 +
                            <div class="i22-line" data-int="int1">
 +
                                <div class="img-cont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/5/58/T--Vilnius-Lithuania--step-2-2-int1.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="letter">A</div>
 +
                                <div class="cn">High</div>
 +
                            </div>
 +
                            <div class="i22-line" data-int="int2">
 +
                                <div class="img-cont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/f0/T--Vilnius-Lithuania--step-2-2-int2.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="letter">B</div>
 +
                                <div class="cn">High</div>
 +
                            </div>
 +
                            <div class="i22-line" data-int="int3">
 +
                                <div class="img-cont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/8/8a/T--Vilnius-Lithuania--step-2-2-int3.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="letter">C</div>
 +
                                <div class="cn">Medium</div>
 +
                            </div>
 +
                            <div class="i22-line" data-int="int4">
 +
                                <div class="img-cont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/35/T--Vilnius-Lithuania--step-2-2-int4.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="letter">D</div>
 +
                                <div class="cn">Low</div>
 +
                            </div>
 +
                            <div class="i22-line" data-int="int5">
 +
                                <div class="img-cont">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/4/44/T--Vilnius-Lithuania--step-2-2-int5.png" width="42"
 +
                                        height="42">
 +
                                </div>
 +
                                <div class="letter">E</div>
 +
                                <div class="cn">Low</div>
 +
                            </div>
 +
                        </div>
 +
                    </div>
 +
                    <div class="right-step">
 +
                        <div class="i23 active">
 +
                            <img src="https://static.igem.org/mediawiki/2017/1/11/T--Vilnius-Lithuania--step-2-3-righthigh.png" width="261" height="128">
 +
                            <span>High</span>
 +
                        </div>
 +
                        <div class="i23">
 +
                            <img src="https://static.igem.org/mediawiki/2017/8/8a/T--Vilnius-Lithuania--step-2-3-rightmedium.png" width="261" height="128">
 +
                            <span>Medium</span>
 +
                        </div>
 +
                        <div class="i23">
 +
                            <img src="https://static.igem.org/mediawiki/2017/3/38/T--Vilnius-Lithuania--step-2-3-rightlow.png" width="261" height="128">
 +
                            <span>Low</span>
 +
                        </div>
 +
                    </div>
 +
                    <div class="right-step"></div>
 +
                    <div class="right-step"></div>
 +
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
    <div class="solution-trigger">
 +
        <div class="notice">Check our future solution! <img src="https://static.igem.org/mediawiki/2017/0/05/T--Vilnius-Lithuania--step-2-5-line.png" width="77" height="52"></div>
 +
        <div class="the-trigger"></div>
 +
        <div class="text">SYNORI</div>
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<h1> Controllable and flexible multi-plasmid system </h1>
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<p>In the field of synthetic biology there is a need for a standardized, stable and quickly expandable <b>multi-plasmid system</b> (A system in which different, unique plasmid groups in could be kept in a cell culture, and no plasmid group would be lost during cell replication). Due to plasmid incompatibility<b>*</b>, current commercially available systems offer up to 5 different plasmids in a cell using different origin of replication (ori) sequences that do not interfere with each other. Yet, these systems are often unstable and unpredictable, as well as limited due to the fact that ori sequences are taken from plasmids that originate from different organisms. It is currently hard to expand the compatible ORI sequence list because we would need to discover those new specific sequences in nature, which would require a considerable amount of effort and time. Also, previously mentioned systems do not offer <b>any</b> way to control plasmid copy number of each plasmid group separately. </p>
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<p>With that in mind we have set ourselves <b>several goals that we want to accomplish in our project</b>: </p>
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<p><li> Development of synthetic plasmid origin of replication (ori) site, based on <a href="https://en.wikipedia.org/wiki/ColE1"> ColE1</a> replicon, which would <b>tackle the problem of incompatibility by using uniquely barcoded RNA I/RNA II gene pairs</b> for each group of plasmid and show that we are able to stably co-maintain at least six of our newly made plasmids in E. coli. Using this strategy we will also gain an opportunity to control the copy number of each plasmid group by adjusting RNA I gene expression of each group separately.</li></p>
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<p><li>Construction of a <b> discrete, recombinase based multi-level synthetic switch system </b> that will allow scientists to precisely control the copy number and ratio between different groups of plasmids, also eliminating the need of periodical addition of inducers to the growth medium.</li></p>
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<p><li>Because our system will contain different plasmids, we are carrying out a research to develop <b>alternatives to the usage of antibiotics</b> (for purposes of biosafety) in order to apply external selection pressure,to ensure that any cell that loses a single group of plasmid would not survive.</li></p>
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<p>In parallel with wet-lab experiments we plan to and currently began <b>writing a mathematical model to describe various aspects of our system</b>. Our dynamic plasmid replication models will allow us to predict theoretical outcomes of the system with various different parameters. We also aim to predict the probabilities of plasmid loss during cell division and parameters that may influence that loss in order to minimize the depletion.</p>
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<p>Our system will open the door to various new applications, such as larger synthetic metabolic pathways, drug synthesis, biological computing and much more!</p>
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<p><b>*</b>Plasmid incompatibility is defined as the inability of two different co-resident plasmids to be stably inherited over a number of generations in the same bacterial cell line. It mainly arises because of the fact that some plasmids share and compete for same replication factors. For example, plasmids that replicate faster due to their small size would outgrow other plasmids really fast in a culture of cells, and the plasmid group with lesser copy number would have a much greater chance to be lost during cell division compared to the dominating group of plasmids.</p>
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Follow and support us at facebook &rarr; <a href="https://www.facebook.com/VilniusiGEM/"><img src="http://i.imgur.com/JF7xQDq.png" alt="Vilnius-Lithuania iGEM facebook" width="29" height=“29"> </a>
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Latest revision as of 03:01, 1 November 2017

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Present Challenges of
Experimenting With Plasmid Systems

1. 1.
Plasmid Replication

Origin of replication (Ori) is a sequence where the plasmid replication begins. Plasmids with similar Ori sites use the same replication machinery.

Problem #1
1. 2.
Unbalanced Replication

Replication machinery does not discriminate between similar Ori sites, therefore smaller plasmids are replicated faster and rapidly outgrow larger plasmids, which leads to uneven plasmid ratios in a cell.

Problem #1
1. 3.
Plasmid Loss

This is even more likely to occur when some plasmids are low copy number. When the cell divides, low copy plasmids tend to be lost in next generations.

Problem #1
2. 1.
Plasmid Incompatibility

This inability to stably co-maintain different plasmids over a number of generations in the same bacterial cell line is called plasmid incompatibility. One needs to use different types of Ori to avoid this trouble.

Problem #2
2. 2.
Plasmid Incompatibility

However, it often causes trouble due to limited Ori selection options or the lack of good combination of Ori and plasmid's copy number. Imagine you require a system consisting of two - low and high copy number plasmids. Try picking them from the list.

Problem #2
2. 3.
Fixed Copy Number

Currently no handy method of changing the copy number of particular plasmid exists. It must be either low, or medium, or high.

Problem #2
2. 4.
Deficient Responsivity

There is also no mechanism to adjust the copy number when so is required in any specific situation, for example: incoming cellular signals or special patterns.

Problem #2

Did this look smart, easy or convenient?

We think it is time for a change.

Plasmid A
Plasmid B
Origin of replication
Replication machinery
Plasmid A
Plasmid B
Origin of replication
Replication machinery
Plasmid A
Plasmid B
Origin of replication
Replication machinery
Different plasmids

A

B

C

D

Different types of ORI
Different replication machinery molecules

Plasmid

Copy number

A
High
B
High
C
Medium
D
Low
E
Low
High
Medium
Low
Check our future solution!
SYNORI