Difference between revisions of "Team:Peking/Demonstrate"

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            <title>Overview</title>
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        <title></title>
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            <meta name="description" content="Wiki of Peking iGEM 2016" />
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        <meta name="description" content="Wiki of Peking iGEM 2016" />
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            <meta name="author" content="Li Jiamian & Wang Yuqing">
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        <meta name="author" content="Li Jiamian & Wang Yuqing"/>
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         </div>
 
         </div>
 
         <!--/Navigation -->
 
         <!--/Navigation -->
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        <!-- Page Title======================================================================== -->
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 +
                    <h1>Results</h1>
 +
                    <p class="title1" style="text-align:center">In this section, you could find what we achieved during this summer. For detailed lab results, models or human practices, please use the navigation bar for a quick webpage-redirecting.</p>
 +
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                                    <h4><a href="javascript:void(0);" onclick="naver('A')">To&nbsp;A</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('B')">To&nbsp;B</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('C')">To&nbsp;C</a></h4>
 +
                                </div>
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                            </div>
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                        </div>
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                            <a id="A"></a>
 +
                            <div class="texttitle">Project Achievements</div>
 +
                            <br/>
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">1.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Constructed a multifunctional protein of interest via molecular biological methods, and introduced the constructed plasmids into the engineered bacteria for protein expression. <a href="https://2016.igem.org/Team:Peking/Basic_Part"/>(Learn more)</a></p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
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                                <div class="info">
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                                    <div class="ordi">2.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Searched for methods and the best conditions for the extraction of each protein. <a href="https://2016.igem.org/Team:Peking/Notebook/Protocol:purification_of_recombinant_proteins"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
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                                <div class="info">
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                                    <div class="ordi">3.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Demonstrated a quick and stable crosslinking process of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher via covalent bonds. We also optimized this reaction concerning the relevant parameters such as temperature, pH, etc.. <a href="https://2016.igem.org/Team:Peking/Crosslinking"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">4.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Demonstrated effective adsorption of uranyl ions by monomeric Triple SpyTag-SUP or polymer network containing the SUP module under a number of conditions. The adsorption was highly efficient and fast, not only under experimental conditions but also in simulated seawater or lake water containing uranium pollution. <a href="https://2016.igem.org/Team:Peking/Uranyl-adsorption"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">5.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Attached biotin to amino-coated magnetic beads and achieved clearance of the polymer network formed via the crosslinking of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher with a magnet. <a href="https://2016.igem.org/Team:Peking/Clearance"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">6.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Set up a signal peptide library and screened for optimally suited signal peptides in order to efficiently secrete the proteins of interest. We found two signal peptides of high efficiency - those derived from OmpA and LtIIb. <a href="https://2016.igem.org/Team:Peking/Secretion"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">7.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Used all the above-mentioned experiments together to demonstrate that the complete Uranium Reaper system, consisting of Triple SpyTag-SUP, Triple SpyTag-mSA, Triple SpyCatcher and biotin-coated magnetic beads, could effectively handle uranium pollution under simulated real-life conditions in about 2 hours. We aimed to optimize this strategy and hoped it could be implemented as a uranyl removal kit. <a href="https://2016.igem.org/Team:Peking/Proof"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">8.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>We exchanged the Triple SpyTag-SUP monomer for Triple SpyTag-LBP or Triple SpyTag-CBP, and tried using the same strategy to adsorb lead and cadmium. The results were remarkable, clearly demonstrating that the Uranium Reaper strategy has much potential to be expanded to other heavy metals. <a href="https://2016.igem.org/Team:Peking/Demonstrate"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                            <br/>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">&nbsp;</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <figure>
 +
                                        <p style="text-align:center;"><img style="width:90%;" src="https://static.igem.org/mediawiki/2016/a/af/T--Peking--image_demotable1.png" alt=""/></p>
 +
                                        <figcaption style="text-align:left;">
 +
                                           
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">&nbsp;</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Even though the efficiency of the Uranium Reaper system may be somewhat lower than current methods, it could certainly be optimized through further development work. Importantly, Uranium Reaper is much better in other aspects. In the future, we plan to optimize the entire Uranium Reaper strategy in order to enhance the adsorption efficiency.</p>
 +
                                </div>
 +
                            </div>
  
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">&nbsp;</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>For an overall view of our project, please redirect to <a href="https://2016.igem.org/Team:Peking/Description"/>Overview Page</a> or <a href="https://2016.igem.org/Team:Peking/Design"/>Design Page</a>. Links to detailed lab results could also be found on these pages.</p>
 +
                                </div>
 +
                            </div>
  
  
−
Demonstrate
+
                           
−
Overview
+
                           
−
The aim of our project is to build a synthetic organelle based on phase separation as a multifunctional platform. Based on the principle of multivalence and interaction, we fused interactional modules into homo-oligomeric tags (HOtags) to form granules in S. cerevisiae. (Click to see more about Background and Design)
+
                            <p>&nbsp;</p>
−
We have built spontaneous and induced synthetic organelles by specific interaction modules, so that we can control the formation process by different ways for demands in biological engineering. Then we characterized the kinetics and properties of synthetic organelles theoretically and experimentally. These results confirm the potential of synthetic organelles in synthetic biology.
+
                           
−
It inspired us to propose some specific applications of our synthetic organelles, including organization hub, sensor, and metabolism regulator. We have verified the feasibility of them by loading GFP-nanobody module, NAD+ sensor module and carotene production module to the whole system.
+
                           
−
We believe that our work has reached the medal requirements of demonstration as we have confirmed that our synthetic organelles can be formed in vivo and deliver a range of functions both for engineering and research due to their amazing properties. The concrete demonstration of the whole platform is shown below. You can see more details of experiments and modeling in our Data_Page and Modeling
+
                           
−
（这里或许需要一张总设计图）
+
                           
−
二、 Phase Separation System
+
                           
−
(一) Spontaneous and induced synthetic organelles can be formed by phase separation
+
                            <a id="B"></a>
−
Our basic system consists of two components of synthetic organelles. Either of them has a specific HOtag to form homo-oligomers. We expect that they are able to form synthetic organelles due to the principles of phase separation. To verify the feasibility of the design, we fused two fluorescence proteins with the two components of synthetic organelles (Figure1.a) so that we can observe the self-organization of components and the formation of granules under fluorescence microscope.
+
                           
−
We used SUMO-SIM interaction module to build a spontaneous organelle. When two components are expressed in yeasts, granules with the two fluorescence proteins can be observed in vivo (Figure1.b).
+
                            <div class="texttitle">Beyond Experiment</div>
−
Meanwhile, by rapamycin induced interaction module, FKBP-Frb, we have built an inducible organelle. We can see granules occurs in yeasts within minutes after adding the inducer.
+
                            <br/>
−
Figure1.a The basic design of synthetic organelles with florescence reporters. （这里可能需要一张cartoon的设计图）
+
                            <div class="coll">
−
            b, c fluorescence images of spontaneous organelles (SUMO-SIM based) and inducible synthetic organelles (FKBP-Frb based, after adding 10000 nM rapamycin)
+
                                <div class="info">
−
 
+
                                    <div class="ordi">1.</div>
−
(二) The formation of organelles has flexible but predictable properties and kinetics in different conditions
+
                                </div>
−
Then we combined modeling of phase separation and experiment to research the kinetics of the organelles formation process expecting that a well-characterized system can reach its whole potential in complex applications.  
+
                                <div class="content">
−
As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications.  
+
                                    <p>We submitted 53 high-quality and well-characterized Standard BioBricks, including a set of derivatives of Triple SpyTag and Triple SpyCatcher, such as the Triple SpyTag-SUP and Triple SpyTag-mSA. <a href="https://2016.igem.org/Team:Peking/Basic_Part"/>(Learn more)</a></p>
−
Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water.
+
                                </div>
−
(b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation.
+
                            </div>
−
(c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.
+
                           
−
 
+
                            <div class="coll">
−
The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.
+
                                <div class="info">
−
Figure3 (a) A simulation of organelle formation process in different interaction strength of components.
+
                                    <div class="ordi">2.</div>
−
(b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.
+
                                </div>
−
 
+
                                <div class="content">
−
We also tried to characterized other properties, like the liquid-like property of the synthetic organelles, as they may affect the functions. See more details about our characterizations in DataPage Phase separation.
+
                                    <p>We developed a special software which could be used to calculate the molecular weight distribution of protein polymers using Flory’s theory. The results of testing have demonstrated that the software is accurate and useful. <a href="https://2016.igem.org/Team:Peking/Software"/>(Learn more)</a> </p>
−
三、 Functional Organelles
+
                                </div>
−
Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.
+
                            </div>
−
Figure4 (organization hub)
+
                           
−
Design of GFP-nanobody based system
+
                            <div class="coll">
−
fluorescence images of GFP-nanobody based system
+
                                <div class="info">
−
Figure5 (sensor)
+
                                    <div class="ordi">3.</div>
−
(a)~(?) fluorescence images of sensor based system
+
                                </div>
−
Figure6 (metabolism)
+
                                <div class="content">
−
Characterization of carotene production system
+
                                    <p>We visited experts from the College of Chemistry and Molecular Engineering and School of Physics of Peking University, respectively, to learn about the current situation surrounding uranium pollution in the real world and how people could control the situation. After finishing the main work, we presented them with the achievements of the project and got their feedback. <a href="https://2016.igem.org/Team:Peking/HP/consulting"/>(Learn more)</a> </p>
−
（phase内和phase外的胡萝卜素生产实验）
+
                                </div>
−
四、 Perspective
+
                            </div>
−
SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.
+
                           
−
 
+
                            <div class="coll">
−
 
+
                                <div class="info">
−
 
+
                                    <div class="ordi">4.</div>
−
 
+
                                </div>
−
 
+
                                <div class="content">
−
            </div> <!-- page-content End-->
+
                                    <p>We did an interview with the Hunan Nuclear Geology 311 Brigade and gained thorough insights into the treatment of uranyl pollution used by the people on the firing line. This way we could compare the methods they were using with the Uranium Reaper strategy. <a href="https://2016.igem.org/Team:Peking/HP/Gold/311"/>(Learn more)</a> </p>
−
        </div> <!-- Content End-->
+
                                </div>
−
       
+
                            </div>
−
       
+
                           
−
       
+
                            <div class="coll">
−
          
+
                                <div class="info">
−
          
+
                                    <div class="ordi">5.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>We helped and collaborated with other iGEM teams by guiding a new team (BHU_China), as well as discussing about project design and technical skills and sharing DNA materials (OUC-China, BIT-China, Tianjin, UCAS, Jinlin_China and BNU-China). <a href="https://2016.igem.org/Team:Peking/Collaborations"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">6.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>We attended the CCiC (Central China iGEM Consortium), which is a large-scale competition-free jamboree of about 50 teams, providing participants with an opportunity for meaningful exchanges of ideas and problem solving. <a href="https://2016.igem.org/Team:Peking/Collaborations"/>(Learn more)</a> </p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                           
 +
                            <p>&nbsp;</p>
 +
                           
 +
                           
 +
                           
 +
                            <a id="C"></a>
 +
                            <div class="texttitle">Our future plan</div>
 +
                            <br/>
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">1.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>We should reproduce all of the experiments that we have done this summer to make sure the results are credible.</p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">2.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>We will optimize the whole strategy to enhance the adsorption efficiency by changing pH, temperature, reaction time of crosslinking and clearance. (The efficiency is only about 60% without further optimization)</p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">3.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>According to the results for the adsorption of 13nM uranyl, the polymer network exhibited a good ability in a simulated seawater environment. We could thus also look into other usage scenarios of Uranium Reaper, such as bio-mining and uranium enrichment.</p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">4.</div>
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>Exchange of the SUP module for other functional proteins. For example, we could integrate proteins which could bind other heavy metals such as mercury so that the polymer network could be used to treat other kinds of pollution as well.</p>
 +
                                </div>
 +
                            </div>
 +
                           
 +
                            <div class="coll">
 +
                                <div class="info">
 +
                                    <div class="ordi">5.</div>
 +
                                </div>
 +
                                <div class="content">
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                                    <p>We could assemble enzyme systems behind the SpyTag backbone to create a production plant in vitro. In the protein polymeric network, the concentration of enzymes could be increased and the efficiency of biocatalysis may consequently also be enhanced.</p>
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                                    <p>If we optimize the number of SpyTag or SpyCatcher modules per protein monomer, as well as the working concentrations of proteins, we may make protein-3D printing using the Spy Crosslinking Network come true.</p>
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Revision as of 07:23, 14 October 2018

Results

In this section, you could find what we achieved during this summer. For detailed lab results, models or human practices, please use the navigation bar for a quick webpage-redirecting.

Project Achievements

1.

Constructed a multifunctional protein of interest via molecular biological methods, and introduced the constructed plasmids into the engineered bacteria for protein expression. (Learn more)

2.

Searched for methods and the best conditions for the extraction of each protein. (Learn more)

3.

Demonstrated a quick and stable crosslinking process of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher via covalent bonds. We also optimized this reaction concerning the relevant parameters such as temperature, pH, etc.. (Learn more)

4.

Demonstrated effective adsorption of uranyl ions by monomeric Triple SpyTag-SUP or polymer network containing the SUP module under a number of conditions. The adsorption was highly efficient and fast, not only under experimental conditions but also in simulated seawater or lake water containing uranium pollution. (Learn more)

5.

Attached biotin to amino-coated magnetic beads and achieved clearance of the polymer network formed via the crosslinking of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher with a magnet. (Learn more)

6.

Set up a signal peptide library and screened for optimally suited signal peptides in order to efficiently secrete the proteins of interest. We found two signal peptides of high efficiency - those derived from OmpA and LtIIb. (Learn more)

7.

Used all the above-mentioned experiments together to demonstrate that the complete Uranium Reaper system, consisting of Triple SpyTag-SUP, Triple SpyTag-mSA, Triple SpyCatcher and biotin-coated magnetic beads, could effectively handle uranium pollution under simulated real-life conditions in about 2 hours. We aimed to optimize this strategy and hoped it could be implemented as a uranyl removal kit. (Learn more)

8.

We exchanged the Triple SpyTag-SUP monomer for Triple SpyTag-LBP or Triple SpyTag-CBP, and tried using the same strategy to adsorb lead and cadmium. The results were remarkable, clearly demonstrating that the Uranium Reaper strategy has much potential to be expanded to other heavy metals. (Learn more)


 

 

Even though the efficiency of the Uranium Reaper system may be somewhat lower than current methods, it could certainly be optimized through further development work. Importantly, Uranium Reaper is much better in other aspects. In the future, we plan to optimize the entire Uranium Reaper strategy in order to enhance the adsorption efficiency.

 

For an overall view of our project, please redirect to Overview Page or Design Page. Links to detailed lab results could also be found on these pages.

 

Beyond Experiment

1.

We submitted 53 high-quality and well-characterized Standard BioBricks, including a set of derivatives of Triple SpyTag and Triple SpyCatcher, such as the Triple SpyTag-SUP and Triple SpyTag-mSA. (Learn more)

2.

We developed a special software which could be used to calculate the molecular weight distribution of protein polymers using Flory’s theory. The results of testing have demonstrated that the software is accurate and useful. (Learn more)

3.

We visited experts from the College of Chemistry and Molecular Engineering and School of Physics of Peking University, respectively, to learn about the current situation surrounding uranium pollution in the real world and how people could control the situation. After finishing the main work, we presented them with the achievements of the project and got their feedback. (Learn more)

4.

We did an interview with the Hunan Nuclear Geology 311 Brigade and gained thorough insights into the treatment of uranyl pollution used by the people on the firing line. This way we could compare the methods they were using with the Uranium Reaper strategy. (Learn more)

5.

We helped and collaborated with other iGEM teams by guiding a new team (BHU_China), as well as discussing about project design and technical skills and sharing DNA materials (OUC-China, BIT-China, Tianjin, UCAS, Jinlin_China and BNU-China). (Learn more)

6.

We attended the CCiC (Central China iGEM Consortium), which is a large-scale competition-free jamboree of about 50 teams, providing participants with an opportunity for meaningful exchanges of ideas and problem solving. (Learn more)

 

Our future plan

1.

We should reproduce all of the experiments that we have done this summer to make sure the results are credible.

2.

We will optimize the whole strategy to enhance the adsorption efficiency by changing pH, temperature, reaction time of crosslinking and clearance. (The efficiency is only about 60% without further optimization)

3.

According to the results for the adsorption of 13nM uranyl, the polymer network exhibited a good ability in a simulated seawater environment. We could thus also look into other usage scenarios of Uranium Reaper, such as bio-mining and uranium enrichment.

4.

Exchange of the SUP module for other functional proteins. For example, we could integrate proteins which could bind other heavy metals such as mercury so that the polymer network could be used to treat other kinds of pollution as well.

5.

We could assemble enzyme systems behind the SpyTag backbone to create a production plant in vitro. In the protein polymeric network, the concentration of enzymes could be increased and the efficiency of biocatalysis may consequently also be enhanced.

6.

If we optimize the number of SpyTag or SpyCatcher modules per protein monomer, as well as the working concentrations of proteins, we may make protein-3D printing using the Spy Crosslinking Network come true.