Difference between revisions of "Team:BGU Israel/Collaborations"

 
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<h2> Collaborations</h2>
 
  
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This year we have collaborated with <a href="https://2015.igem.org/Team:Stockholm#">Team Stockholm</a> in human cells expression of an Affibody which presents itself on the plasma membrane. This Affibody could then, for example, bind a toxin or a fluorescent molecule, which will kill or color the cells expressing it.
 
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Although the collaboration was such that had expressed the Affibody in human cells, without the use of our design, when using our system, specifically our third-plasmid design, the Affibody could be expressed solely in cancerous cells, and introduction of the desired molecule would have an effect only in diseased tissue.
 
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        <h2>Collaboration</h2><br />
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            <p>We have collaborated with several iGEM teams this year:</p> <br/>
 +
            <ul>
 +
                <li><a href="#Stockholm">Scientific collaborations -Stockholm</a> </li>
 +
                <li><a href="#Amoy">Written articles for the Amoy newsletter</a></li>
 +
                <li><a href="#Aix">Collaboration on human practice survey</a></li>
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                <li><a href="#Technion">Attended "Mini jamboree" conference hosted by the "Technion"</a></li>
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            </ul>
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 +
           
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            <a name="Stockholm"></a>
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            <h4>Introduction</h4><br />
 +
            <p><a href="https://2015.igem.org/Team:Stockholm"> Stockholm team </a>is working on cancer diagnosis by engineering of a bacterial sensor which could detect trace amounts of cancer biomarkers.</p>
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                <br/>    <br/>    <br/>
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                    <td>
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                        <h5>Stockholm Abstract:</h5><br/>
 +
                        <p>"Synthetic biology offers many potential cost-effective healthcare technologies. One of those could be new ways to diagnose and treat disease at an early stage. Current techniques for biomarker detection (e.g. ELISA, RIA) are time consuming, expensive and require specialised equipment.</p>
 +
                        <p>We intend to design a microbiological system for the detection of low quantities of biomarkers. This assay aims to be easier and more cost efficient than existing techniques and possible to perform in modestly equipped settings. Initially, we will focus on the expression of a receptor for the desired biomarker. Depending on the nature of the biomarker, the receptor will be either be native or designed. </p>
 +
                        <p>Upon biomarker detection, signal amplification will be triggered by our receptor system to activate a read out/detection system (e.g. Luciferase, GFP) inside the microorganism to artificially amplify the extracellular signal. In the next stage, the team will go on to design a read-out system to measure the concentration of biomarkers in body samples. Finally we want to investigate if we can make this system transferable to other biomarkers, changing only the receptor part of the system. </p>
 +
                        <p>The system would be cheap, fast and possible to distribute without advanced equipment. Our motivation is to improve patient prognosis and quality of life and to improve efficiency and reduce costs within the healthcare system." </p>
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            <br/>
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            <p>Our assistance to the team is to express a GPI-anchored Affibody <a href="http://parts.igem.org/Part:BBa_K1766012">[BBa_K1766012]</a>  on the plasma membrane of human cancer cells, which can then be specifically targeted by a compatible drug or toxin (Fig. 1).
 +
            </p><br/>
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            <div align="center"><img src="https://static.igem.org/mediawiki/2015/4/47/BGU_Sstockholm_Collaboration.PNG" width="910px" height="400" /><br/>
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            Fig. 1. Possible applications of Affibody expression in cancer cells.</div>
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            <br/><br/>
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            <h4>Results</h4><br/>
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            <h5>1. Cloning</h5><br/>
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            <p>We cloned the Affibody <a href="http://parts.igem.org/Part:BBa_K1766012">[BBa_K1766012]</a> into our AAV vector for expression under CMV promoter (Fig. 2).
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                      <td width="80%" style="border-style: hidden;"><div align="left"><b>A</b></div></td>
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                    <td width="20%" style="border-style: hidden;"><div align="left"><b>B</b></div></td>
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                    <td width="80%" style="border-style: hidden;"><img src="https://static.igem.org/mediawiki/2015/b/bd/Collaboration_cloning_result1.png" height="500px" width="600px"/></td>
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                    <td width="20%" style="border-style: hidden;"><a href="https://static.igem.org/mediawiki/2015/3/37/Collaboration_cloning_result2.png" target="blank"><img src="https://static.igem.org/mediawiki/2015/3/37/Collaboration_cloning_result2.png" height="200px" width="150px"/></a></td>
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            <br/> Fig. 2. A. Cloning map of Affibody into AAV vector. B. Successful cloning results.
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            <h5> 2. Affibody expression in human cancer cells</h5><br/>
 +
            Affibody-AAV was produced <a href="https://static.igem.org/mediawiki/2015/d/dd/AAV_production_Protocol.pdf">[link to protocol of virus production]</a>, and used for transduction of human cancer cell line (HT1080 fibrosarcoma cell line).<br/>
 +
            As the Affibody was predesigned to include 3xFLAG peptide (DYKDDDDK) in its sequence, we used anti-FLAG-specific primary antibody to specifically detect the protein in human cancer cells (HT1080 fibrosarcoma) using immunofluorescence <a href="https://static.igem.org/mediawiki/2015/7/73/IF_staining_protocol.pdf">[link to staining protocol]</a>.
 +
We used two staining protocols, with and w/o permeabilization step, in an attempt to distinguish between extracellular membrane localization and intracellular expression. As shown in Fig. 3, transfection with Affibody-expressing vector resulted in detectable Affibody expression only when permeabilization step was applied. This could indicate the the protein construct is expressed, but it is not correctly transported and localized to the outer membrane, or FLAG sequence (the antigen for the primary Ab) is located inside the cytoplasm.
 +
 
 +
<br/><br/>
 +
<img src="https://static.igem.org/mediawiki/2015/6/6c/Affibody-ST.jpg">
 +
            <br/> Fig. 3. Affibody expression in human cancer cells. A-B. Representative images of Affibody expression in human fibrosarcoma cell line HT1080, using Alexa-488-conjugated secondary antibody (methanol fixation & permeabilization). C. Lack of staining w/o permeabilization step (fixation with 4% PFA in PBS). D. Negative control (w/o primary antibody).
 +
           
 +
     
 +
            <h4>Conclusion and future aspects</h4>
 +
 
 +
            <p>Affibody construct could be successfully expressed in human cancer cells. However, the design of the construct should be optimized to ensure the expression on the outer cell membrane for its subsequent use for cancer cell tagging and targeting. For detection, primary Ab against Affibody part of the construct is preferable, in order to clearly validate the expression on the outer membrane.
 +
                In the future, the Affibody could be expressed exclusively in cancer cells using our Boomerang approach using dCas9-VP64-gRNA system under the control of cancer-specific promoters.
 +
            </p>
 +
 
 +
            <br/> <br/>
 +
            <h2>Polices & Practices collaborations:</h2>
 +
           
 +
            <a name="France"></a>
 +
            <h5>France-Aix-Maseille, Questioner</h5>
 +
                <a name="Aix"></a>
 +
            <p>Aix-Marseille Université (France), proposed a collaboration. They needed to collect a lot of information about chewing-gum and GMOs around the world.
 +
                They prepared a small survey, and we went to the streets and interviewed random people.
 +
                It was a fun challenge. We received a gold badge for our help.  </p>
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                            <h5>Aix-Marseille project idea:</h5><br/>
 +
                            <p>"Nowadays, chewing gum is the second urban pollutant after cigarette butts. To clean chewing gum, specific engines are used but they are heavy and expensive. The time required for its use is long and they need a large quantity of water. Besides economical aspects, chewing gums have an environmental impact. They are dangerous for the wildlife such as birds. Indeed, believing that it is bread, they eat chewing gums and choke. Regarding these alarming facts, we have decided to create a new environmentally- responsible way to sanitize our streets." </p>
 +
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 +
            <a name="Amoy"></a>
 +
            <h5>China-Amoy university, article</h5>
 +
                    <p>Amoy iGEM team issued monthly newsletter that enabled igem teams from all over the world to share information about their project and discuss issues concerning it and synthetic biology in general.
 +
                      For this year’s version of the Newsletter iGEM Amoy contacted iGEM  Ben-Gurion to include us in the 2015 issue.  We had the honor to collaborate and publish an article about our project in the fifth issue of 2015 Newsletter. We described  our project and an update on our progress.</p>
 +
            <a href="https://static.igem.org/mediawiki/2015/1/1f/2015-Newsletter-No.5.pdf" target="blank">link to our article</a> <br/>
 +
           
 +
 
 +
            <a name="Technion"></a>
 +
            <h5>Israel-Technion, Mini Jamboree</h5><br/><p>On September 9th, We were invited along with the other Israeli iGEM teams to the Technion - Mini Jamboree Event.
 +
Here, each team presented their project, and a panel of judges asked questions and gave tips for improvement and better preparation for the Giant Jamboree in Boston.</p>
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Latest revision as of 09:22, 18 September 2015

Team:BGU Israel



Collaboration


We have collaborated with several iGEM teams this year:


Introduction


Stockholm team is working on cancer diagnosis by engineering of a bacterial sensor which could detect trace amounts of cancer biomarkers.




Stockholm Abstract:

"Synthetic biology offers many potential cost-effective healthcare technologies. One of those could be new ways to diagnose and treat disease at an early stage. Current techniques for biomarker detection (e.g. ELISA, RIA) are time consuming, expensive and require specialised equipment.

We intend to design a microbiological system for the detection of low quantities of biomarkers. This assay aims to be easier and more cost efficient than existing techniques and possible to perform in modestly equipped settings. Initially, we will focus on the expression of a receptor for the desired biomarker. Depending on the nature of the biomarker, the receptor will be either be native or designed.

Upon biomarker detection, signal amplification will be triggered by our receptor system to activate a read out/detection system (e.g. Luciferase, GFP) inside the microorganism to artificially amplify the extracellular signal. In the next stage, the team will go on to design a read-out system to measure the concentration of biomarkers in body samples. Finally we want to investigate if we can make this system transferable to other biomarkers, changing only the receptor part of the system.

The system would be cheap, fast and possible to distribute without advanced equipment. Our motivation is to improve patient prognosis and quality of life and to improve efficiency and reduce costs within the healthcare system."


Our assistance to the team is to express a GPI-anchored Affibody [BBa_K1766012] on the plasma membrane of human cancer cells, which can then be specifically targeted by a compatible drug or toxin (Fig. 1).



Fig. 1. Possible applications of Affibody expression in cancer cells.


Results


1. Cloning

We cloned the Affibody [BBa_K1766012] into our AAV vector for expression under CMV promoter (Fig. 2).

A
B

Fig. 2. A. Cloning map of Affibody into AAV vector. B. Successful cloning results.
2. Affibody expression in human cancer cells

Affibody-AAV was produced [link to protocol of virus production], and used for transduction of human cancer cell line (HT1080 fibrosarcoma cell line).
As the Affibody was predesigned to include 3xFLAG peptide (DYKDDDDK) in its sequence, we used anti-FLAG-specific primary antibody to specifically detect the protein in human cancer cells (HT1080 fibrosarcoma) using immunofluorescence [link to staining protocol]. We used two staining protocols, with and w/o permeabilization step, in an attempt to distinguish between extracellular membrane localization and intracellular expression. As shown in Fig. 3, transfection with Affibody-expressing vector resulted in detectable Affibody expression only when permeabilization step was applied. This could indicate the the protein construct is expressed, but it is not correctly transported and localized to the outer membrane, or FLAG sequence (the antigen for the primary Ab) is located inside the cytoplasm.


Fig. 3. Affibody expression in human cancer cells. A-B. Representative images of Affibody expression in human fibrosarcoma cell line HT1080, using Alexa-488-conjugated secondary antibody (methanol fixation & permeabilization). C. Lack of staining w/o permeabilization step (fixation with 4% PFA in PBS). D. Negative control (w/o primary antibody).

Conclusion and future aspects

Affibody construct could be successfully expressed in human cancer cells. However, the design of the construct should be optimized to ensure the expression on the outer cell membrane for its subsequent use for cancer cell tagging and targeting. For detection, primary Ab against Affibody part of the construct is preferable, in order to clearly validate the expression on the outer membrane. In the future, the Affibody could be expressed exclusively in cancer cells using our Boomerang approach using dCas9-VP64-gRNA system under the control of cancer-specific promoters.



Polices & Practices collaborations:

France-Aix-Maseille, Questioner

Aix-Marseille Université (France), proposed a collaboration. They needed to collect a lot of information about chewing-gum and GMOs around the world. They prepared a small survey, and we went to the streets and interviewed random people. It was a fun challenge. We received a gold badge for our help.

Aix-Marseille project idea:

"Nowadays, chewing gum is the second urban pollutant after cigarette butts. To clean chewing gum, specific engines are used but they are heavy and expensive. The time required for its use is long and they need a large quantity of water. Besides economical aspects, chewing gums have an environmental impact. They are dangerous for the wildlife such as birds. Indeed, believing that it is bread, they eat chewing gums and choke. Regarding these alarming facts, we have decided to create a new environmentally- responsible way to sanitize our streets."

China-Amoy university, article

Amoy iGEM team issued monthly newsletter that enabled igem teams from all over the world to share information about their project and discuss issues concerning it and synthetic biology in general. For this year’s version of the Newsletter iGEM Amoy contacted iGEM Ben-Gurion to include us in the 2015 issue. We had the honor to collaborate and publish an article about our project in the fifth issue of 2015 Newsletter. We described our project and an update on our progress.

link to our article
Israel-Technion, Mini Jamboree

On September 9th, We were invited along with the other Israeli iGEM teams to the Technion - Mini Jamboree Event. Here, each team presented their project, and a panel of judges asked questions and gave tips for improvement and better preparation for the Giant Jamboree in Boston.