Difference between revisions of "Team:BIT-China/Design"
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background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)"> | background: url(https://static.igem.org/mediawiki/2016/4/45/T--BIT-China--content_bg.jpg)"> | ||
| − | + | <div class="content-right" style="float: left;width:100%;padding: 10px;"> | |
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| − | + | <img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--content_decoration.png" | |
| − | + | alt="content_decoration" style="position:absolute;right: 0px;height: 150px;;top: 10px;"> | |
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| − | + | <div class="block-border-outer" id="content-right-border"> | |
| − | + | <div class="overview-content clearfix"> | |
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<div class="content-title col-sm-12"> | <div class="content-title col-sm-12"> | ||
<img src="https://static.igem.org/mediawiki/2016/3/30/T--BIT-China--Project--Design--title.png" | <img src="https://static.igem.org/mediawiki/2016/3/30/T--BIT-China--Project--Design--title.png" | ||
alt="title" class="col-sm-8 col-sm-offset-2"> | alt="title" class="col-sm-8 col-sm-offset-2"> | ||
| − | |||
</div> | </div> | ||
<!--问题描述--> | <!--问题描述--> | ||
| − | <div class="problem-txt col-sm-12"> | + | <div class="problem-txt block-content col-sm-12"> |
<div class="problem-title">What is the problem?</div> | <div class="problem-title">What is the problem?</div> | ||
<div> | <div> | ||
<i class="fa fa-thumb-tack" aria-hidden="true"></i> | <i class="fa fa-thumb-tack" aria-hidden="true"></i> | ||
| − | <span>Plasmid segragational instability has been the limit step for large-scale protein production and bioremediation. | + | <span>Plasmid segragational instability has been the limit step for large-scale protein production and bioremediation. Antibiotics for plasmid retention is not practical in this situation.</span> |
</div> | </div> | ||
<div> | <div> | ||
<i class="fa fa-thumb-tack" aria-hidden="true"></i> | <i class="fa fa-thumb-tack" aria-hidden="true"></i> | ||
| − | <span>High copy number ( | + | <span>High copy number (HCN) plasmids are lost from population at a high rate due to the large metabolic burden and lack of nature factors for plasmid maintenance.</span> |
</div> | </div> | ||
</div> | </div> | ||
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<div> | <div> | ||
| − | Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller), we need to select a signal which can indicate the intracellular plasmid numbers. There is a basic rule: when the plasmid numbers are above | + | Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller), and we need to select a signal which can indicate the intracellular plasmid numbers. Here we give a definition, when the bacteria containing few plasmids consume lots of nutrition but don’t work efficiently, we called them slackers. There is a basic rule: when the plasmid numbers are above a threshold, we regard the bacterium as normally-working and the P-SLACKiller won't start; however, when the plasmid numbers are below the threshold, we judge it as a slacker and the P-SLACKiller will kill these slackers, so that we can achieve the goal of increasing the plasmid maintenance. In the end, in order to kill the slackers and thus control the plasmid numbers, we selected the inhibitor protein as the signal molecular and the in-promoter repressed by inhibitor as a switch to start the killer gene. |
</div> | </div> | ||
</div> | </div> | ||
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<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig1.png" | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig1.png" | ||
| − | alt="fig1" class="center-block" style="width: | + | alt="fig1" class="center-block" style="width:100%;"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.1</b> The basic circuit of P-SLACKiller.</div> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | We use the constitutive promoter to express the inhibitor protein, thus the intracellular inhibitor concentration is positively correlated with the plasmid numbers. The inhibitor can repress the in-promoter and control the expression of downstream killer gene. To avoid the unexpected | + | <br>We use the constitutive promoter to express the inhibitor protein, thus the intracellular inhibitor concentration is positively correlated with the plasmid numbers. So the inhibitor can be used as a signal indicating the plasmid numbers. The inhibitor can repress the in-promoter and control the expression of downstream killer gene. When the inhibitor concentration reduces to a threshold, the downstream killer gene will express. To avoid the unexpected leak out of killer gene caused by the constitutive promoter, we designed the gene circuit with the constitutive promoter and the in-promoter transcribing along different directions like Fig.1. |
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
The genetic locus of functional gene can be substituted on the basis of production demand in the future. | The genetic locus of functional gene can be substituted on the basis of production demand in the future. | ||
</div> | </div> | ||
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</div> | </div> | ||
<div> | <div> | ||
| − | Under normal circumstance | + | Under normal circumstance, the plasmid numbers are above the threshold, the inhibitor concentration is high enough to completely repress the activity of in-promoter. Considering the effect of plasmids segregational instability, the concentration of plasmids in some cells will decline with time going on. With no intervention, these cells will gradually become slackers as well as the predominant strain among the population. In our project, when the plasmid numbers are below the threshold, the decrease of inhibitor proteins will lead to the increase of in-promoter activity. At this time, the killer gene will express and kill the cell. We define that, when the killer expressed is just able to kill the cell, the plasmid numbers is the threshold we've talked about. Based on this, we concluded that when the plasmid numbers are above the threshold, the bacteria can survive and have high working efficiency. This range is called survival range. To prevent the slackers consume a lot of nutrition, we kill it when the range removes to the dead one. |
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | In this way, we can optimize the general population structure through plasmid-numbers- | + | In this way, we can optimize the general population structure through plasmid-numbers-maintenance. |
</div> | </div> | ||
| − | <div> | + | <div><div> |
<img src="https://static.igem.org/mediawiki/2016/0/01/T--BIT-China--Project--Design--fig2.png" | <img src="https://static.igem.org/mediawiki/2016/0/01/T--BIT-China--Project--Design--fig2.png" | ||
| − | alt=" | + | alt="fig2" class="center-block" style="width:100%"></div> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.2</b> With the decrease of plasmid numbers, the concentration of repressor will reduce to a threshold. According, in-promoter activity will rise up and star the transcription of killer gene. </div> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
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<div class="block-content-brief"> | <div class="block-content-brief"> | ||
<div> | <div> | ||
| − | The most critical factor in our system is the inhibitor concentration since it controls the switch of our killer system. Thus we need to prove that the in-promoter will have different responses corresponding to different concentrations of | + | The most critical factor in our system is the inhibitor concentration since it controls the switch of our killer system. Thus we need to prove that the in-promoter will have different responses corresponding to different concentrations of inhibitor. |
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | We combined the wet experiment results | + | We combined the wet experiment results with the mathematical model to measure the final result. |
| − | <a href="https://2016.igem.org/Team:BIT-China/Model" style="color: blue;"> | + | <a href="https://2016.igem.org/Team:BIT-China/Model" style="color: blue;"> [know more about our model]</a> |
</div> | </div> | ||
| − | <div class="step-style"> | + | <div class="step-style block-header-margin1"> |
<i class="fa fa-play-circle" aria-hidden="true"></i> | <i class="fa fa-play-circle" aria-hidden="true"></i> | ||
First Step | First Step | ||
</div> | </div> | ||
<div> | <div> | ||
| − | Since | + | Since it's hard to control the plasmid numbers, we employed the arabinose induced P<sub>BAD</sub></b> promoter. The plasmid numbers is stable in short time. By adding different concentration of arabinose, we can simulate the production of inhibitors with different concentration. In this way, we can see the downstream in-promoter stay in the corresponding statues. <span class="italic">RFP</span> is used to replace the killer gene for simplifying our wet experiment. To expand the application of our system, we choose two combinations of inhibitor and in-promoter. |
</div> | </div> | ||
</div> | </div> | ||
| − | <div class="content-block-item"> | + | <div> |
| + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | ||
| + | <span class="block-content-header">Goal</span> | ||
| + | </div> | ||
| + | <div class="content-block-item" style="margin: 0px auto;padding: 0 1em;"> | ||
<div> | <div> | ||
| − | + | (1) Build two parallel circuits employing different inhibitor proteins | |
| − | + | ||
</div> | </div> | ||
<div> | <div> | ||
| − | + | (2) Find the relationship between the inhibitor and in-promoter | |
| − | + | ||
| − | + | ||
| − | (2) Find the relationship | + | |
</div> | </div> | ||
<div> | <div> | ||
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</div> | </div> | ||
</div> | </div> | ||
| − | <div | + | <div> |
| − | + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | |
| − | + | <span class="block-content-header">Part Design</span> | |
| − | + | </div> | |
| − | + | <div class="content-block-item"style="margin: 0px auto;padding: 0 1em;"> | |
<div> | <div> | ||
The gene circuits are shown in Fig.3. | The gene circuits are shown in Fig.3. | ||
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<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig3.png" | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--BIT-China--Project--Design--fig3.png" | ||
| − | alt="fig3" class="center-block" style="width: 80%;"> | + | alt="fig3" class="center-block" style="width:80%"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.3</b> The gene circuits of threshold device. </div> | ||
| + | |||
</div> | </div> | ||
<div> | <div> | ||
| − | (1) <b>Part:BBa_K808000 | + | <br>(1) <b>Part:BBa_K808000 <span class="italic">AraC</span>-P<sub>BAD</sub></b> is used to control the expression of inhibitor proteins, The concentration of inducer arabinose is regarded as an input signal, and the red fluorescence intensity controlled by in-promoter is the output. |
</div> | </div> | ||
<div> | <div> | ||
| − | (2) | + | (2) Two combinations of inhibitor and in-promoter are selected to prove the concept of plasmid quorum sensing. Due to the limit of time, we measured 1-2 circuits. |
| − | <a href="https://2016.igem.org/Team:BIT-China/Inhibitor" | + | <a href="https://2016.igem.org/Team:BIT-China/Inhibitor" |
| + | target="_blank" style="color: blue;">[see inhibitor mechanisms]</a> | ||
</div> | </div> | ||
<div> | <div> | ||
| − | (3) RFP is used as an indicator since | + | (3) <span class="italic">RFP</span> is used as an indicator since it's easier to quantify compared with the killer gene. |
</div> | </div> | ||
<div> | <div> | ||
| − | (4) The inducible promoter | + | (4) The inducible promoter P<sub>BAD</sub></b> and the in-promoter express towards different directions, a terminator B0015 is added between the two promoters. |
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div> | <div> | ||
| − | Different concentrations of arabinose will induce inhibitor with different | + | Different concentrations of arabinose will induce inhibitor with different concentration of inhibitors. |
</div> | </div> | ||
<div> | <div> | ||
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</div> | </div> | ||
<div> | <div> | ||
| − | By adding different concentration of arabinose, we can measure the fluorescence intensity of RFP and find out the relationship | + | By adding different concentration of arabinose, we can measure the fluorescence intensity of RFP and find out the relationship between the arabinose and RFP intensity and the initial thresholds under the control of certain inhibitor proteins. |
</div> | </div> | ||
</div> | </div> | ||
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<!--second--> | <!--second--> | ||
<div class="block-content-brief"> | <div class="block-content-brief"> | ||
| − | <div class="step-style"> | + | <div class="step-style block-header-margin1"> |
<i class="fa fa-play-circle" aria-hidden="true"></i> | <i class="fa fa-play-circle" aria-hidden="true"></i> | ||
Second step | Second step | ||
</div> | </div> | ||
<div> | <div> | ||
| − | In order to find out the concentrations of inhibitor under different concentrations of arabinose and use the results to derive the threshold of plasmids number in the constitutive circuit. Inhibitor is replaced by RFP and induced based on the same condition. The concentration of inhibitor can represented by measuring the fluorescence intensity of RFP. | + | In order to find out the concentrations of inhibitor under different concentrations of arabinose and use the results to derive the threshold of plasmids number in the constitutive circuit. Inhibitor is replaced by RFP and induced based on the same condition. The concentration of inhibitor can be represented by measuring the fluorescence intensity of RFP. |
</div> | </div> | ||
</div> | </div> | ||
| − | <div class="content-block-item"> | + | <div> |
| + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | ||
| + | <span class="block-content-header">Goal</span> | ||
| + | </div> | ||
| + | <div class="content-block-item"style="margin: 0px auto;padding: 0 1em;"> | ||
<div> | <div> | ||
| − | + | (1) Represent the concentration of inhibitor by measuring the fluorescence intensity of RFP. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | (1)Represent the concentration of inhibitor by measuring the fluorescence intensity of RFP. | + | |
</div> | </div> | ||
<div> | <div> | ||
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<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/b/b4/T--BIT-China--Project--Design--fig4.png" | <img src="https://static.igem.org/mediawiki/2016/b/b4/T--BIT-China--Project--Design--fig4.png" | ||
| − | alt="fig4" class="center-block" style="width: | + | alt="fig4" class="center-block" style="width: 60%;"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"> | ||
| + | <b>Fig.4</b> The gene circuit constructed to find out the concentrations of inhibitor induced by different concentrations of arabinose. </div> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
<div class="block-content-item"> | <div class="block-content-item"> | ||
<div> | <div> | ||
| − | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | + | <br><i class="fa fa-hand-o-right" aria-hidden="true"></i> |
<span class="block-content-header">We assume that:</span> | <span class="block-content-header">We assume that:</span> | ||
</div> | </div> | ||
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<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/7/7e/T--BIT-China--Project--Design--fig5.png" | <img src="https://static.igem.org/mediawiki/2016/7/7e/T--BIT-China--Project--Design--fig5.png" | ||
| − | alt="fig5" style="width: 80%;"> | + | alt="fig5" style="width: 80%;"class="center-block"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.5</b> The relationship between the concentrations of inhibitor and plasmid number. </div> | ||
| + | |||
</div> | </div> | ||
<div> | <div> | ||
| − | Thus we could find out the relationship between the concentration of arabinose and inhibitor. | + | <br>Thus we could find out the relationship between the concentration of arabinose and inhibitor. |
</div> | </div> | ||
</div> | </div> | ||
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<!--third--> | <!--third--> | ||
<div class="block-content-brief"> | <div class="block-content-brief"> | ||
| − | <div class="step-style"> | + | <div class="step-style block-header-margin1"> |
<i class="fa fa-play-circle" aria-hidden="true"></i> | <i class="fa fa-play-circle" aria-hidden="true"></i> | ||
Third step | Third step | ||
</div> | </div> | ||
<div> | <div> | ||
| − | We use plasmids with different copy numbers to simulate the concentration of plasmids losing on different levels. Three kinds of plasmids, pSB1k3(100~200 copies), | + | We use plasmids with different copy numbers to simulate the concentration of plasmids losing on different levels. Three kinds of plasmids, <span class="italic">pSB1k3</span> (100~200 copies), <span class="italic">pSB3k3</span> (20~30 copies), <span class="italic">pSB4k5</span> (5~10 copies), are chosen and constructed with target fragment. By observing our system's working conditions, we can finally prove its function of controlling plasmids number. |
</div> | </div> | ||
</div> | </div> | ||
| − | <div | + | <div> |
| − | + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | |
| − | + | <span class="block-content-header">Goal</span> | |
| − | + | </div> | |
| − | + | <div class="content-block-item" style="margin: 0px auto;padding: 0 1em;"> | |
<div> | <div> | ||
(1) Simulate the plasmids losing on different levels | (1) Simulate the plasmids losing on different levels | ||
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<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--Project--Design--fig6.png" | <img src="https://static.igem.org/mediawiki/2016/5/5c/T--BIT-China--Project--Design--fig6.png" | ||
| − | alt="fig6" class="center-block" style="width: | + | alt="fig6" class="center-block" style="width: 60%;"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.6</b> The gene circuits of threshold device with constitutive promoter. </div> | ||
| + | |||
</div> | </div> | ||
| − | <div> | + | <div style="margin: 10px auto;padding: 0 1em;"> |
(1) We constructed the circuit on different plasmids with different copy numbers. | (1) We constructed the circuit on different plasmids with different copy numbers. | ||
| − | + | <br>(2) The locus of constitutive promoter will be replaced by another constitutive promoter which has the similar strength as P<sub>BAD</sub></b> promoter. | |
| − | + | ||
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div> | <div> | ||
| − | When the number of plasmids is less than N, inhibitor is not strong enough to inhibit the in-promoter totally, so the killer gene will express triggering the death of bacteria. On the contrary, if the number of plasmids is higher than N, the bacteria | + | When the number of plasmids is less than N, inhibitor is not strong enough to inhibit the in-promoter totally, so the killer gene will express triggering the death of bacteria. On the contrary, if the number of plasmids is higher than N, the bacteria won't die. |
</div> | </div> | ||
<div> | <div> | ||
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<!--fourth--> | <!--fourth--> | ||
<div class="block-content-brief"> | <div class="block-content-brief"> | ||
| − | <div class="step-style"> | + | <div class="step-style block-header-margin1"> |
<i class="fa fa-play-circle" aria-hidden="true"></i> | <i class="fa fa-play-circle" aria-hidden="true"></i> | ||
Forth step | Forth step | ||
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</div> | </div> | ||
</div> | </div> | ||
| − | <div | + | <div> |
| − | + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | |
| − | + | <span class="block-content-header">Goal</span> | |
| − | + | </div> | |
| − | + | <div class="content-block-item" style="margin: 0px auto;padding: 0 1em;"> | |
<div> | <div> | ||
(1) Adjust the threshold of plasmids by substituting different biobricks. | (1) Adjust the threshold of plasmids by substituting different biobricks. | ||
| Line 401: | Line 410: | ||
The gene circuits are shown in Fig.7. | The gene circuits are shown in Fig.7. | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
<img src="https://static.igem.org/mediawiki/2016/c/cd/T--BIT-China--Project--Design--fig7.png" | <img src="https://static.igem.org/mediawiki/2016/c/cd/T--BIT-China--Project--Design--fig7.png" | ||
alt="fig7" class="center-block" style="width: 80%;"> | alt="fig7" class="center-block" style="width: 80%;"> | ||
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.7</b> The gene circuits of threshold device constructed on a different plasmids with different copy numbers. </div> | ||
| + | |||
</div> | </div> | ||
| − | <div> | + | <div style="margin: 10px auto;padding: 0 1em;"> |
(1) Simulate the plasmids losing on different levels. | (1) Simulate the plasmids losing on different levels. | ||
| − | + | <br>(2) The locus of B0034 and J23119 will be replaced by B0032 and J23109 J23116. | |
| − | + | ||
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="block-content-brief"> | <div class="block-content-brief"> | ||
<div> | <div> | ||
| − | As an executor for killing the cell labeled as slackers, killer device should include the detecting part like in-promoter interacting with inhibitor protein as well as the killing part like toxin gene mazF and hokD. | + | As an executor for killing the cell labeled as slackers, killer device should include the detecting part like in-promoter interacting with inhibitor protein as well as the killing part like toxin gene <span class="italic">mazF</span> and <span class="italic">hokD</span>. |
</div> | </div> | ||
</div> | </div> | ||
<div class="block-content-item"> | <div class="block-content-item"> | ||
| − | <div | + | <div> |
| − | + | <i class="fa fa-hand-o-right" aria-hidden="true"></i> | |
| − | + | <span class="block-content-header">Ways of self-killing:</span> | |
| − | + | </div> | |
| − | + | <div class="block-content-item-block" style="margin: 0px auto;padding: 0 1em;"> | |
<div> | <div> | ||
| − | 1) toxic protein | + | 1) toxic protein |
</div> | </div> | ||
<div> | <div> | ||
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</div> | </div> | ||
<div> | <div> | ||
| − | At the first stage, we need to verify the function of two toxin proteins and make sure the bacteria can be killed after the expression of toxin proteins. To control the expression of killer gene, instead of J23119, arabinose inducible regulatory promoter/repressor unit | + | At the first stage, we need to verify the function of two toxin proteins and make sure the bacteria can be killed after the expression of toxin proteins. To control the expression of killer gene, instead of J23119, arabinose inducible regulatory promoter/repressor unit is employed in our circuit. |
</div> | </div> | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/2/2a/T--BIT-China--Project--Design--fig8.png" | <img src="https://static.igem.org/mediawiki/2016/2/2a/T--BIT-China--Project--Design--fig8.png" | ||
| − | alt="fig8" class="center-block" style="width: 80%;"> | + | alt="fig8" class="center-block" style="width:80%;"> |
| + | <div class="center-block" style="font-size:0.9em;text-align:center"><b>Fig.8</b> Circuits for toxin gene function testing through induction by arabinose. </div> | ||
| + | |||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | After measuring the growth curve, we observed the obvious difference between the testing group and control group. | + | <br>After measuring the growth curve, we observed the obvious difference between the testing group and control group. |
| − | <a href="https://2016.igem.org/Team:BIT-China/Results">[see results page for killer]</a> | + | <a href="https://2016.igem.org/Team:BIT-China/Results" target="_blank" style="color: blue;">[see results page for killer]</a> |
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with the killer circuit and replace the RFP for killer gene. | + | To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with the killer circuit and replace the <span class="italic">RFP</span> for killer gene. |
| − | <a href="#threshold_device">[see design for threshold]</a> | + | <a href="#threshold_device" style="color: blue;">[see design for threshold]</a> |
</div> | </div> | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/0/08/T--BIT-China--Project--Design--fig9.png" | <img src="https://static.igem.org/mediawiki/2016/0/08/T--BIT-China--Project--Design--fig9.png" | ||
| − | alt="fig9" class="center-block" style="width: | + | alt="fig9" class="center-block" style="width:85%;"> |
| + | <div class="pic_info"><b>Fig.9</b> Circuits for testing for killing capacity under different concentration of arabinose, which can be regard as under different plasmid concentrations. </div> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | For the toxic protein, MazF and HokD were chosen as candidates. | + | <br>For the toxic protein, MazF and HokD were chosen as candidates. |
| − | <a href="https://2016.igem.org/Team:BIT-China/Killing_Mechanisms">[see more about the killing mechanisms]</a> | + | <a href="https://2016.igem.org/Team:BIT-China/Killing_Mechanisms" target="_blank" style="color: blue;">[see more about the killing mechanisms]</a> |
</div> | </div> | ||
</div> | </div> | ||
| Line 480: | Line 492: | ||
<div id="upgraded_p-slackiller" class=" block-title">Upgraded P-SLACKiller</div> | <div id="upgraded_p-slackiller" class=" block-title">Upgraded P-SLACKiller</div> | ||
<!--Upgraded P-SLACKiller 问题描述--> | <!--Upgraded P-SLACKiller 问题描述--> | ||
| − | <div class="problem-txt"> | + | <div class="problem-txt block-content"> |
| − | <div class="problem-title">Why do we need to optimize our project?</div> | + | <div class="problem-title" style="text-align:left">Why do we need to optimize our project?</div> |
<div> | <div> | ||
<!--<img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"--> | <!--<img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"--> | ||
| − | + | <!--alt="ul_logo1" width="18" height="18">--> | |
| − | + | <span> | |
| − | + | It's possible to produce generation of plasmid-free cells, even if all plasmids were losen. Thus, we decided to integrate the killer device into genome to solve this problem. | |
| − | + | <a href="https://2016.igem.org/Team:BIT-China/Recombination" target="_blank" style="color: blue;">[see reombination]</a> | |
| − | + | </span> | |
</div> | </div> | ||
<div> | <div> | ||
<!--<img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"--> | <!--<img src="https://static.igem.org/mediawiki/2016/c/c1/T--BIT-China--Project--Design--ul_logo1.png"--> | ||
| − | + | <!--alt="ul_logo1" width="18" height="18">--> | |
<span> | <span> | ||
To control the plasmid number above different thresholds, we need to adjust the initial threshold through replacement of RBS as well as in-promoter mutation. | To control the plasmid number above different thresholds, we need to adjust the initial threshold through replacement of RBS as well as in-promoter mutation. | ||
| − | + | <a href="https://2016.igem.org/Team:BIT-China/Promoter" target="_blank" style="color: blue;">[see mutation]</a> | |
</span> | </span> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
<div id="recombination" class="block-title">Recombination</div> | <div id="recombination" class="block-title">Recombination</div> | ||
<div class="block-content"> | <div class="block-content"> | ||
<div class="block-content-brief"> | <div class="block-content-brief"> | ||
| − | <div> | + | <div style="text-align:left"> |
Recombineering ( | Recombineering ( | ||
<b>recombi</b>nation-mediated genetic engi<b>neering</b>) is an efficient molecular | <b>recombi</b>nation-mediated genetic engi<b>neering</b>) is an efficient molecular | ||
engineering technique used for gene replacement, deletion and insertion. | engineering technique used for gene replacement, deletion and insertion. | ||
</div> | </div> | ||
| − | |||
</div> | </div> | ||
| − | |||
<div class="content-block-item"> | <div class="content-block-item"> | ||
| Line 516: | Line 527: | ||
<span class="block-content-header">Ways: a linear plus circular reaction</span> | <span class="block-content-header">Ways: a linear plus circular reaction</span> | ||
</div> | </div> | ||
| − | <div> | + | <div style="margin: 0px auto;padding: 0 1em;"> |
| − | A：Employment of traditional λ-Red recombination | + | A：Employment of traditional λ-Red recombination |
</div> | </div> | ||
</div> | </div> | ||
| Line 531: | Line 542: | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/9/96/T--BIT-China--Project--Design--fig10.png" | <img src="https://static.igem.org/mediawiki/2016/9/96/T--BIT-China--Project--Design--fig10.png" | ||
| − | alt="fig10" style="width: | + | alt="fig10" style="width:100%;" class="center-block"> |
| + | <div class="pic_info"><b>Fig.10</b> The basic circuit of upgraded P-SLACKiller. </div> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | Compared with the original one, two homologous arms (50bp) are added. With the help of lambda Red recombination system, the killer part can be integrated into genome, preventing the whole system from being invalid. The final gene circuit is shown in Fig.11. | + | <br>Compared with the original one, two homologous arms (50bp) are added. With the help of lambda Red recombination system, the killer part can be integrated into genome, preventing the whole system from being invalid. The final gene circuit is shown in Fig.11. |
</div> | </div> | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/d/de/T--BIT-China--Project--Design--fig11.png" | <img src="https://static.igem.org/mediawiki/2016/d/de/T--BIT-China--Project--Design--fig11.png" | ||
| − | alt="fig11" style="width: | + | alt="fig11" style="width:100%;" class="center-block"> |
| + | <div class="pic_info"><b>Fig.11</b> The basic circuit of upgraded P-SLACKiller. </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 544: | Line 557: | ||
<div class="content-block-item"> | <div class="content-block-item"> | ||
<div> | <div> | ||
| − | B: CRISPR-Cas9 coupling λ-Red recombineering | + | <br>B: CRISPR-Cas9 coupling λ-Red recombineering |
</div> | </div> | ||
</div> | </div> | ||
| Line 555: | Line 568: | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/7/74/T--BIT-China--Project--Design--fig12.png" | <img src="https://static.igem.org/mediawiki/2016/7/74/T--BIT-China--Project--Design--fig12.png" | ||
| − | alt="fig12" style="width: | + | alt="fig12" style="width:100%;" class="center-block"> |
| + | <div class="pic_info"><b>Fig.12</b> Double plasmid to meet different requirements for each element. Equip the high copy number plasmid PUC19 with sgRNA targeting site on genome and donor fragment with homologous arms. The low copy number plasmid are equipped with expression cassette of cas9 protein and λ-Red recombinase. </div> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | Difference between the two methods above: Traditional way use the resistance gene as a selection marker, while the CRISPR coupled way introduce a double-strand break (DSB) into the strain with no recombination occurred. | + | <br>Difference between the two methods above: Traditional way use the resistance gene as a selection marker, while the CRISPR coupled way introduce a double-strand break (DSB) into the strain with no recombination occurred. |
</div> | </div> | ||
</div> | </div> | ||
| Line 570: | Line 584: | ||
In our project, we tried both the two ways to test the integration efficiency. However, the CRISPR coupled way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction. | In our project, we tried both the two ways to test the integration efficiency. However, the CRISPR coupled way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction. | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | We tried the traditional way with the pKD46 plasmid provided by Chun | + | We tried the traditional way with the <span class="italic">pKD46</span> plasmid provided by Chun Li's lab and got the positive results. |
<a href="https://2016.igem.org/Team:BIT-China/Results" style="color: blue;">[results for recombination]</a> | <a href="https://2016.igem.org/Team:BIT-China/Results" style="color: blue;">[results for recombination]</a> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
Two main factors will influence the recombination efficiency: the target site on genome and the length of homologous arms between donor fragment. | Two main factors will influence the recombination efficiency: the target site on genome and the length of homologous arms between donor fragment. | ||
| − | <a href="https://2016.igem.org/Team:BIT-China/Protocol">[recombination protocol]</a> | + | <a href="https://2016.igem.org/Team:BIT-China/Protocol" style="color: blue;">[recombination protocol]</a> |
</div> | </div> | ||
</div> | </div> | ||
| Line 585: | Line 599: | ||
<div class="block-content-brief"> | <div class="block-content-brief"> | ||
For inductive promoters, two kinds of region are significant to its transcription strength. One is common -10 and -35 region which can firmly bind to RNA polymerase, the other is operon region binding with regulatory factors like inhibitor proteins. | For inductive promoters, two kinds of region are significant to its transcription strength. One is common -10 and -35 region which can firmly bind to RNA polymerase, the other is operon region binding with regulatory factors like inhibitor proteins. | ||
| − | <a href="" style="color: blue;">[see mutation mechanisms]</a> | + | <a href="https://2016.igem.org/Team:BIT-China/Promoter" target="_blank" style="color: blue;">[see mutation mechanisms]</a> |
</div> | </div> | ||
| Line 592: | Line 606: | ||
<span class="block-content-header"> Aim:</span> | <span class="block-content-header"> Aim:</span> | ||
</div> | </div> | ||
| − | <div> | + | <div style="margin: 0px auto;padding: 0 1em;"> |
| − | (1) To adjust threshold through changing in- | + | (1) To adjust threshold through changing in-promoter's strength |
| − | + | <br>(2) Tied to specific promoter like P<sub>Tet</sub></b>, build a mutants library and improve the existing part by measuring the strength through RFP | |
| − | + | ||
| − | + | ||
</div> | </div> | ||
<div> | <div> | ||
| Line 604: | Line 616: | ||
<div> | <div> | ||
<img src="https://static.igem.org/mediawiki/2016/d/d2/T--BIT-China--Project--Design--fig13.png" | <img src="https://static.igem.org/mediawiki/2016/d/d2/T--BIT-China--Project--Design--fig13.png" | ||
| − | alt="fig9" class="center-block" style="width: | + | alt="fig9" class="center-block" style="width: 50%;"> |
| + | <div class="pic_info"><b>Fig.13</b> The basic circuit of promoter mutation. </div> | ||
</div> | </div> | ||
| − | <div> | + | <div class="block-paragraph"> |
| − | After analyzing the promoter region of | + | <br>After analyzing the promoter region of P<sub>Tet</sub></b>, we designed random primers to mutate its -35 region. RFP intensity is measured to indicate the strength of mutated promoters. |
<a href="" style="color: blue;">[see mutation results]</a> | <a href="" style="color: blue;">[see mutation results]</a> | ||
</div> | </div> | ||
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</div> | </div> | ||
| − | <div class="nav-left" id="nav_left_wrapper" | + | <div class="slow-transition nav-left" id="nav_left_wrapper"> |
| + | |||
| + | <div class="nav_shrink" id="nav_shrink"> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/8/8a/T--BIT-China--img--pumpkin--collapse.png" alt="nav_shrink_icon" | ||
| + | id="nav_shrink_icon" width="60"> | ||
| + | </div> | ||
<div class="nav-left-title" style="position:relative;width: 200px;padding: 0;"> | <div class="nav-left-title" style="position:relative;width: 200px;padding: 0;"> | ||
<img src="https://static.igem.org/mediawiki/2016/4/43/T--BIT-China--img--nav_left_top.png" alt="nav_left_top" style="width: 100%;"> | <img src="https://static.igem.org/mediawiki/2016/4/43/T--BIT-China--img--nav_left_top.png" alt="nav_left_top" style="width: 100%;"> | ||
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</div> | </div> | ||
| − | + | <!--相邻页面切换按钮--> | |
| − | <!--相邻页面切换按钮--> | + | <div id="site-change-btn" |
| − | <div | + | style="position: absolute;left: 20px;bottom: 20px;z-index:9;display: none;"> |
| − | + | <a href="https://2016.igem.org/Team:BIT-China/Background"> | |
| − | + | ||
<span style="color: #654D6F;font-weight: bold;font-size: 12px"> | <span style="color: #654D6F;font-weight: bold;font-size: 12px"> | ||
| − | <i class="fa fa-angle-double-left" aria-hidden="true"></i> | + | <i class="fa fa-angle-double-left" aria-hidden="true"></i>Background</span> |
| − | + | </a> | |
| − | + | <img src="https://static.igem.org/mediawiki/2016/2/2c/T--BIT-China--img--page_change.png" | |
| − | + | alt="page_change" style="width: 40px;height: auto;"> | |
| − | + | <a href="https://2016.igem.org/Team:BIT-China/Results"> | |
<span style="color: #654D6F;font-weight: bold;font-size: 12px"> | <span style="color: #654D6F;font-weight: bold;font-size: 12px"> | ||
Results<i class="fa fa-angle-double-right" aria-hidden="true"></i></span> | Results<i class="fa fa-angle-double-right" aria-hidden="true"></i></span> | ||
| − | + | </a> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 02:39, 11 November 2016
What is the problem?
Plasmid segragational instability has been the limit step for large-scale protein production and bioremediation. Antibiotics for plasmid retention is not practical in this situation.
High copy number (HCN) plasmids are lost from population at a high rate due to the large metabolic burden and lack of nature factors for plasmid maintenance.
Our P-SLACKiller
IDEA
Equip the bacteria with a plasmid-sensing logically adjustable cell killer (P-SLACKiller), and we need to select a signal which can indicate the intracellular plasmid numbers. Here we give a definition, when the bacteria containing few plasmids consume lots of nutrition but don’t work efficiently, we called them slackers. There is a basic rule: when the plasmid numbers are above a threshold, we regard the bacterium as normally-working and the P-SLACKiller won't start; however, when the plasmid numbers are below the threshold, we judge it as a slacker and the P-SLACKiller will kill these slackers, so that we can achieve the goal of increasing the plasmid maintenance. In the end, in order to kill the slackers and thus control the plasmid numbers, we selected the inhibitor protein as the signal molecular and the in-promoter repressed by inhibitor as a switch to start the killer gene.
PART DESIGN
The basic circuit is shown in Fig.1.
Fig.1 The basic circuit of P-SLACKiller.
We use the constitutive promoter to express the inhibitor protein, thus the intracellular inhibitor concentration is positively correlated with the plasmid numbers. So the inhibitor can be used as a signal indicating the plasmid numbers. The inhibitor can repress the in-promoter and control the expression of downstream killer gene. When the inhibitor concentration reduces to a threshold, the downstream killer gene will express. To avoid the unexpected leak out of killer gene caused by the constitutive promoter, we designed the gene circuit with the constitutive promoter and the in-promoter transcribing along different directions like Fig.1.
The genetic locus of functional gene can be substituted on the basis of production demand in the future.
INSIDE MECHANISM
Under normal circumstance, the plasmid numbers are above the threshold, the inhibitor concentration is high enough to completely repress the activity of in-promoter. Considering the effect of plasmids segregational instability, the concentration of plasmids in some cells will decline with time going on. With no intervention, these cells will gradually become slackers as well as the predominant strain among the population. In our project, when the plasmid numbers are below the threshold, the decrease of inhibitor proteins will lead to the increase of in-promoter activity. At this time, the killer gene will express and kill the cell. We define that, when the killer expressed is just able to kill the cell, the plasmid numbers is the threshold we've talked about. Based on this, we concluded that when the plasmid numbers are above the threshold, the bacteria can survive and have high working efficiency. This range is called survival range. To prevent the slackers consume a lot of nutrition, we kill it when the range removes to the dead one.
In this way, we can optimize the general population structure through plasmid-numbers-maintenance.
Fig.2 With the decrease of plasmid numbers, the concentration of repressor will reduce to a threshold. According, in-promoter activity will rise up and star the transcription of killer gene.
Threshold device
The most critical factor in our system is the inhibitor concentration since it controls the switch of our killer system. Thus we need to prove that the in-promoter will have different responses corresponding to different concentrations of inhibitor.
We combined the wet experiment results with the mathematical model to measure the final result.
[know more about our model]
First Step
Since it's hard to control the plasmid numbers, we employed the arabinose induced PBAD promoter. The plasmid numbers is stable in short time. By adding different concentration of arabinose, we can simulate the production of inhibitors with different concentration. In this way, we can see the downstream in-promoter stay in the corresponding statues. RFP is used to replace the killer gene for simplifying our wet experiment. To expand the application of our system, we choose two combinations of inhibitor and in-promoter.
Goal
(1) Build two parallel circuits employing different inhibitor proteins
(2) Find the relationship between the inhibitor and in-promoter
(3) Find out the initial thresholds by measuring RFP intensity
(4) Provide experimental data for modeling part
Part Design
The gene circuits are shown in Fig.3.
Fig.3 The gene circuits of threshold device.
(1) Part:BBa_K808000 AraC-PBAD is used to control the expression of inhibitor proteins, The concentration of inducer arabinose is regarded as an input signal, and the red fluorescence intensity controlled by in-promoter is the output.
(2) Two combinations of inhibitor and in-promoter are selected to prove the concept of plasmid quorum sensing. Due to the limit of time, we measured 1-2 circuits.
[see inhibitor mechanisms]
(3) RFP is used as an indicator since it's easier to quantify compared with the killer gene.
(4) The inducible promoter PBAD and the in-promoter express towards different directions, a terminator B0015 is added between the two promoters.
We assume that:
Different concentrations of arabinose will induce inhibitor with different concentration of inhibitors.
The high concentration of inducers can simulate high concentration of plasmids due to the same effect of producing more inhibitor proteins.
By adding different concentration of arabinose, we can measure the fluorescence intensity of RFP and find out the relationship between the arabinose and RFP intensity and the initial thresholds under the control of certain inhibitor proteins.
Second step
In order to find out the concentrations of inhibitor under different concentrations of arabinose and use the results to derive the threshold of plasmids number in the constitutive circuit. Inhibitor is replaced by RFP and induced based on the same condition. The concentration of inhibitor can be represented by measuring the fluorescence intensity of RFP.
Goal
(1) Represent the concentration of inhibitor by measuring the fluorescence intensity of RFP.
(2) Derive the threshold of plasmids number in the constitutive circuit
Part Design
The gene circuits are shown in Fig.4.
Fig.4 The gene circuit constructed to find out the concentrations of inhibitor induced by different concentrations of arabinose.
We assume that:
More arabinose added, more RFP expressed which can stand for the concentration of inhibitor, and vice versa.
Fig.5 The relationship between the concentrations of inhibitor and plasmid number.
Thus we could find out the relationship between the concentration of arabinose and inhibitor.
Third step
We use plasmids with different copy numbers to simulate the concentration of plasmids losing on different levels. Three kinds of plasmids, pSB1k3 (100~200 copies), pSB3k3 (20~30 copies), pSB4k5 (5~10 copies), are chosen and constructed with target fragment. By observing our system's working conditions, we can finally prove its function of controlling plasmids number.
Goal
(1) Simulate the plasmids losing on different levels
(2) Prove that our system is functional
Part Design
The gene circuits are shown in Fig.6.
Fig.6 The gene circuits of threshold device with constitutive promoter.
(1) We constructed the circuit on different plasmids with different copy numbers.
(2) The locus of constitutive promoter will be replaced by another constitutive promoter which has the similar strength as PBAD promoter.
(2) The locus of constitutive promoter will be replaced by another constitutive promoter which has the similar strength as PBAD promoter.
We assume that:
When the number of plasmids is less than N, inhibitor is not strong enough to inhibit the in-promoter totally, so the killer gene will express triggering the death of bacteria. On the contrary, if the number of plasmids is higher than N, the bacteria won't die.
We expect that in the high copy number plasmid circuit, fluorescence gene will express, and in the low copy number plasmid circuit, there will no fluorescence.
Forth step
The threshold of plasmid number is decided by the relationship between inhibitor and in-promoter. To achievement the goal of build a controllable system, we decide to use different RBS (B0034 B0032) and promoters (J23119 J23116 J23109) with different strengths to change their concentrations. Thus we can finally change the threshold of plasmid as we hope.
Goal
(1) Adjust the threshold of plasmids by substituting different biobricks.
Part Design
The gene circuits are shown in Fig.7.
Fig.7 The gene circuits of threshold device constructed on a different plasmids with different copy numbers.
(1) Simulate the plasmids losing on different levels.
(2) The locus of B0034 and J23119 will be replaced by B0032 and J23109 J23116.
(2) The locus of B0034 and J23119 will be replaced by B0032 and J23109 J23116.
Killer device
As an executor for killing the cell labeled as slackers, killer device should include the detecting part like in-promoter interacting with inhibitor protein as well as the killing part like toxin gene mazF and hokD.
Ways of self-killing:
1) toxic protein
2) sgRNA targeting genome of strains with no NHEJ repair system
3) sgRNA targeting essential genes
PART DESIGN
At the first stage, we need to verify the function of two toxin proteins and make sure the bacteria can be killed after the expression of toxin proteins. To control the expression of killer gene, instead of J23119, arabinose inducible regulatory promoter/repressor unit is employed in our circuit.
Fig.8 Circuits for toxin gene function testing through induction by arabinose.
After measuring the growth curve, we observed the obvious difference between the testing group and control group. [see results page for killer]
To facilitate the measurement of plasmid threshold, we combined the inhibitor circuit with the killer circuit and replace the RFP for killer gene.
[see design for threshold]
Fig.9 Circuits for testing for killing capacity under different concentration of arabinose, which can be regard as under different plasmid concentrations.
For the toxic protein, MazF and HokD were chosen as candidates. [see more about the killing mechanisms]
Upgraded P-SLACKiller
Why do we need to optimize our project?
It's possible to produce generation of plasmid-free cells, even if all plasmids were losen. Thus, we decided to integrate the killer device into genome to solve this problem.
[see reombination]
To control the plasmid number above different thresholds, we need to adjust the initial threshold through replacement of RBS as well as in-promoter mutation.
[see mutation]
Recombination
Recombineering (
recombination-mediated genetic engineering) is an efficient molecular
engineering technique used for gene replacement, deletion and insertion.
Ways: a linear plus circular reaction
A：Employment of traditional λ-Red recombination
PART DESIGN
The basic circuit of upgraded P-SLACKiller is shown in Fig.10.
Fig.10 The basic circuit of upgraded P-SLACKiller.
Compared with the original one, two homologous arms (50bp) are added. With the help of lambda Red recombination system, the killer part can be integrated into genome, preventing the whole system from being invalid. The final gene circuit is shown in Fig.11.
Fig.11 The basic circuit of upgraded P-SLACKiller.
B: CRISPR-Cas9 coupling λ-Red recombineering
PART DESIGN
Fig.12 Double plasmid to meet different requirements for each element. Equip the high copy number plasmid PUC19 with sgRNA targeting site on genome and donor fragment with homologous arms. The low copy number plasmid are equipped with expression cassette of cas9 protein and λ-Red recombinase.
Difference between the two methods above: Traditional way use the resistance gene as a selection marker, while the CRISPR coupled way introduce a double-strand break (DSB) into the strain with no recombination occurred.
Application:
In our project, we tried both the two ways to test the integration efficiency. However, the CRISPR coupled way are deleted from our plan list due to the failure of cas9 and λ-Red plasmid construction.
We tried the traditional way with the pKD46 plasmid provided by Chun Li's lab and got the positive results.
[results for recombination]
Two main factors will influence the recombination efficiency: the target site on genome and the length of homologous arms between donor fragment.
[recombination protocol]
Promoter mutation
For inductive promoters, two kinds of region are significant to its transcription strength. One is common -10 and -35 region which can firmly bind to RNA polymerase, the other is operon region binding with regulatory factors like inhibitor proteins.
[see mutation mechanisms]
Aim:
(1) To adjust threshold through changing in-promoter's strength
(2) Tied to specific promoter like PTet, build a mutants library and improve the existing part by measuring the strength through RFP
(2) Tied to specific promoter like PTet, build a mutants library and improve the existing part by measuring the strength through RFP
Part Design
Fig.13 The basic circuit of promoter mutation.
After analyzing the promoter region of PTet, we designed random primers to mutate its -35 region. RFP intensity is measured to indicate the strength of mutated promoters. [see mutation results]
