Difference between revisions of "Team:CGU Taiwan/Entrepreneurship"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Awards#SpecialPrizes">Best Supporting Entrepreneurship award</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<h2>Entrepreneurship</h2>
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In previous years, iGEM had an entrepreneurship track. Teams were encouraged to build projects and focus around commercializing their work. We have now moved to an award as the best work in this area may come from teams who are not solely focused on entrepreneurship.
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<p>The Best Supporting Entrepreneurship award recognizes exceptional effort to build a business case and commercialize an iGEM project. This award is open to all teams to show that entrepreneurship is something all teams can aspire to do with their project. This award can go to an new project, or to a previous project that a team aimed to commercialize. Have you filed a provisional patent on your project/device/process? Have you raised money to build and ship products? Have you pitched your idea to investors and received money? Complete the entrepreneurship section on the 2016 Judging form and tell us what you did. As always in iGEM, the aim is to impress the judges! </p>
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<h5>Inspiration</h5>
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<p>You can look at what other teams did to get some inspiration! <br />
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Here are a few examples:</p>
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<li><a href="https://2014.igem.org/Team:UCC_Ireland">2014 UCC Ireland</a></li>
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<li><a href="https://2014.igem.org/Team:Imperial">2014 Imperial College London</a></li>
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<li><a href="https://2013.igem.org/Team:UCL_E">2013 University College London Entrepreneurship</a></li>
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<li><a href="https://2012e.igem.org/wiki/index.php/Team:MIT_E">2012 MIT Entrepreneurship</a></li>
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<img alt="" style="position:absolute;z-index:++1;float:left;margin-top:20px;width:390px;height:80px;"  src="https://static.igem.org/mediawiki/2016/b/bb/CGU_Taiwan--logo2.jpg">
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<img style="position:absolute;left:1170px;top:35px;z-index:+1;width:155px;height:180px;" src="https://static.igem.org/mediawiki/2016/8/8a/CGU_Taiwan--logo9.jpg">
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<h1 class="headline">Leijuvant <small></small></h1>
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<ul class="header-subnav">
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan">HOME</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Achievements">ACHIEVEMENTS</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Description">PROJECT</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Software">MODELING</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Human_Practices">HUMAN PRACTICES</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Team">PEOPLE</a></li>
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<li class="dropdown"><a href="https://2016.igem.org/Team:CGU_Taiwan/Interlab">INTERLAB</a></li>
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<li class="dropdown"><a href="https://2016.igem.org/Team:CGU_Taiwan/Safety">SAFETY</a></li>
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<li><a href="https://2016.igem.org/Team:CGU_Taiwan/Parts">PARTS</a></li>
 
</ul>
 
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</header>
  
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<div class="mid" style="top:180px;">
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<br>
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<div style="font-size:60px;color:#9999CC;text-decoration:none;">
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Entrepreneurship
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</div>
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<br><br>
  
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<b>(1) Short introduction of our project</b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Transgenic Leishmania that can be inactivated by light exposure acts as a safe carrier to deliver specific antigens to the APCs for T cells and humoral response. Based on this concept, we established a new model system to generate antigen-specific inactive Leishmania adjuvant (Leijuvant).
 
</div>
 
</div>
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<br><br>
  
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<b>(2) What organisms do we use? </b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Leishmania, DC2.4, E.coli(DH5α, BL21), C57BL/6 mice
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</div>
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<br><br>
  
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<b>(3) Which part do we use? </b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Full length of HA and OVA: We used full length sequence of hemagglutinin (HA) of H1N1 and Ovalbumin as our testing antigen that is expressed in Leishmania. <br>
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5’UTR, 3’UTR, 2.3kb intron: the sequence is originally from Leishmania genome. Act as promotor, terminator, and expression modulating part in our device. 
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</div>
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<br><br>
  
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<b>(4) Where do we perform the experiments?</b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Leishmania and DC2.4 culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. Once the Leishmania is inactivated it can be manipulated on the open bench. DH5α and BL21 are manipulated in the Laminar flow. Normal experiments are performed on open bench with gloves.
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</div>
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<br><br>
  
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<b>(5) The training of the lab team</b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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We received lecture of the following topics: registration for the use of biological materials, human pathogens, recombinant DNA experiments, potentially infectious materials, animals handling, the use of biological safety cabinets (BSCs) and other laminar flow benches (LFBs), emergency procedures for incidents, biosafety levels signs and labels, etc. We further received laboratory practices training of biosafety level 1 and 2. All the experiments are under Dr. Yu and Dr. Chang’s instructions.
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</div>
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<br><br>
  
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<b>(6) How do we protect us? </b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Follow the requirements from different biosafety level laboratory strictly, including wearing gloves, lab coats, masks and goggles if required.
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</div>
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<br><br>
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<b>(7) Safety concern in our project</b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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Leishmania is a genus of trypanosomes that are responsible for the disease leishmaniasis, a blood-borne disease that is spread by sandflies. Leishmania culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. There is almost no chance of infecting people under this experimental condition since there is no direct contact with Leishmania. Also, Leishmania will not be able to survive once it leaves the culture condition. Once it is photo-inactivated it can be manipulated on the open bench.
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</div>
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<br><br>
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<b>(8) How to verify the safety of leishmania after photo-inactivation</b>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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1. How to conduct the photo-inactivation?
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
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Photosensitizers (PS) can be delivered endogenously with delta-aminolevulinate (ALA) and exogenously with phthalocyanines (PC).
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Detailed protocol (link)<br>
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With loading ALA, we can see the florescence with the excitation of uroporphyrin in the 12-well tissue culture plate during illumination of long-wave UV lamp (365nm)1(Fig. 1A.). There wasn’t any illumination by UV lamp without loading ALA(Fig. 1B.). After illuminating with long-wave  UV ， After long-wave UV illumination, we then put it under the LED red light with the wavelength of 620~630nm for 10 minutes (Fig. 1C.) and the double photo inactivation is complete. <br><br>
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<img src="https://static.igem.org/mediawiki/2016/f/f1/LeishSafety1.png" width=550px height=400px style="border:2px black solid;border-radius:8px;"></img>
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</div>
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<br><br>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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2. How to confirm the sucess of photo-inactivation?<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<font size=2>According to (Chang, K. P., et al. 2016.)2we can use the following method to confirm the safety of photo-inactivated Leishmania.</font>
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
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<b> (I) Microscopy of flagellar motility</b>
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
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After illuminating by long-wave UV and red light, you can observe the samples under the microscope. The difference between [+drug, +light]; [+drug, -light]; [-drug;-light] groups can be easily seen. With the illumination of the both chemicals, all six wells of one plate show no flagellar motility under 100x magnification. However, the control group, [+drug, -light] and [-drug;-light], have high motility same as the wild-type Leishmania.
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
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<b>(II) Cultivation for growth (2 weeks)</b>
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
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To further confirm the complete inactivation of Leishmania, we separated 100μl from each sample, [+drug, +light], [+drug, -light] and [-drug;-light], right after illumination. And add them into 24-Well plate containing M199+10%HI-FB medium. Observed and kept tract of it continuously for two weeks. Except the [+drug, +light] group, the other two groups were moving and increasing normally. As for the [+drug, +light] group, there was no motility, or proliferation of the Leishmania from the beginning to the end of the two weeks.
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</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:120px;">
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<b> (III) 2 months after subcutaneously injecting inactivated Leishmania into mice</b>
 +
</div>
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<div style="color:black;text-decoration:none;font-size:18px;margin-left:170px;">
 +
We subcutaneously injected mouse with108 of photo-inactivated Leishmania on both sides of the back around the shoulders, and injected normal saline as the control group. There was no skin ulcers or inflammation and no any other Leishmaniasis symptoms among the 10 mouse that serve as our experimental group(Fig. 2.). <br><br>
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<img src="https://static.igem.org/mediawiki/2016/6/6d/LeishSafety2.png" width=550px height=400px style="border:2px black solid;border-radius:8px;"></img>
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</div>
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<br>
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<div style="color:black;text-decoration:none;font-size:12px;margin-left:170px;">
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Reference:<br>
 +
1. Dutta, S., et al. (2008). "Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin." Eukaryot Cell 7(7): 1146-1157.<br>
 +
2. Chang, K. P., et al. (2016). "New "light" for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives." Parasit Vectors 9(1): 396.
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</div>
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<br><br>
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 +
<b>(9) The product safety concern in the future</b>
 +
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
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<u>Environmental concern</u><br>
 +
Leishmania culture requires a certain osmolarity and combination of nutrients. Therefore, our transgenic Leishmania will not survive as soon as it leaves the medium.<br>
 +
<u>Side effects of adjuvant </u><br>
 +
There are some potential adverse effects underlying every drug and vaccine. More animal test should be carried out and the most efficient dose in human will be selected before clinical trials. In the clinical study, side effect after immunization will be identified and study design will be modified as well. If our product passes the FDA approval, the report of unexpected effect will be always under inspection.<br>
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</div>
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<br><br>
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 +
<b>(10) How would your project be used in the real world?</b>
 +
<div style="color:black;text-decoration:none;font-size:18px;margin-left:70px;">
 +
Our Leijuvant can act to facilitate both humeral and cellular immune response which may contribute to the development of vaccine nowadays. Combined with our prediction software, the platform can predict the peptides on MHC molecules and help to optimize the peptide presentation which may benefit to engineer the sequence for further research and developing a more efficient vaccine.
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<br><br>
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Revision as of 23:26, 19 October 2016

Leijuvant


Entrepreneurship


(1) Short introduction of our project
Transgenic Leishmania that can be inactivated by light exposure acts as a safe carrier to deliver specific antigens to the APCs for T cells and humoral response. Based on this concept, we established a new model system to generate antigen-specific inactive Leishmania adjuvant (Leijuvant).


(2) What organisms do we use?
Leishmania, DC2.4, E.coli(DH5α, BL21), C57BL/6 mice


(3) Which part do we use?
Full length of HA and OVA: We used full length sequence of hemagglutinin (HA) of H1N1 and Ovalbumin as our testing antigen that is expressed in Leishmania.
5’UTR, 3’UTR, 2.3kb intron: the sequence is originally from Leishmania genome. Act as promotor, terminator, and expression modulating part in our device.


(4) Where do we perform the experiments?
Leishmania and DC2.4 culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. Once the Leishmania is inactivated it can be manipulated on the open bench. DH5α and BL21 are manipulated in the Laminar flow. Normal experiments are performed on open bench with gloves.


(5) The training of the lab team
We received lecture of the following topics: registration for the use of biological materials, human pathogens, recombinant DNA experiments, potentially infectious materials, animals handling, the use of biological safety cabinets (BSCs) and other laminar flow benches (LFBs), emergency procedures for incidents, biosafety levels signs and labels, etc. We further received laboratory practices training of biosafety level 1 and 2. All the experiments are under Dr. Yu and Dr. Chang’s instructions.


(6) How do we protect us?
Follow the requirements from different biosafety level laboratory strictly, including wearing gloves, lab coats, masks and goggles if required.


(7) Safety concern in our project
Leishmania is a genus of trypanosomes that are responsible for the disease leishmaniasis, a blood-borne disease that is spread by sandflies. Leishmania culturing and experiments will be performed in a BSL-2 lab within the flow hood in an independent room and requires wearing lab gloves. There is almost no chance of infecting people under this experimental condition since there is no direct contact with Leishmania. Also, Leishmania will not be able to survive once it leaves the culture condition. Once it is photo-inactivated it can be manipulated on the open bench.


(8) How to verify the safety of leishmania after photo-inactivation
1. How to conduct the photo-inactivation?
Photosensitizers (PS) can be delivered endogenously with delta-aminolevulinate (ALA) and exogenously with phthalocyanines (PC). Detailed protocol (link)
With loading ALA, we can see the florescence with the excitation of uroporphyrin in the 12-well tissue culture plate during illumination of long-wave UV lamp (365nm)1(Fig. 1A.). There wasn’t any illumination by UV lamp without loading ALA(Fig. 1B.). After illuminating with long-wave UV ， After long-wave UV illumination, we then put it under the LED red light with the wavelength of 620~630nm for 10 minutes (Fig. 1C.) and the double photo inactivation is complete.



2. How to confirm the sucess of photo-inactivation?
     According to (Chang, K. P., et al. 2016.)2we can use the following method to confirm the safety of photo-inactivated Leishmania.
(I) Microscopy of flagellar motility
After illuminating by long-wave UV and red light, you can observe the samples under the microscope. The difference between [+drug, +light]; [+drug, -light]; [-drug;-light] groups can be easily seen. With the illumination of the both chemicals, all six wells of one plate show no flagellar motility under 100x magnification. However, the control group, [+drug, -light] and [-drug;-light], have high motility same as the wild-type Leishmania.
(II) Cultivation for growth (2 weeks)
To further confirm the complete inactivation of Leishmania, we separated 100μl from each sample, [+drug, +light], [+drug, -light] and [-drug;-light], right after illumination. And add them into 24-Well plate containing M199+10%HI-FB medium. Observed and kept tract of it continuously for two weeks. Except the [+drug, +light] group, the other two groups were moving and increasing normally. As for the [+drug, +light] group, there was no motility, or proliferation of the Leishmania from the beginning to the end of the two weeks.
(III) 2 months after subcutaneously injecting inactivated Leishmania into mice
We subcutaneously injected mouse with108 of photo-inactivated Leishmania on both sides of the back around the shoulders, and injected normal saline as the control group. There was no skin ulcers or inflammation and no any other Leishmaniasis symptoms among the 10 mouse that serve as our experimental group(Fig. 2.).


Reference:
1. Dutta, S., et al. (2008). "Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin." Eukaryot Cell 7(7): 1146-1157.
2. Chang, K. P., et al. (2016). "New "light" for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives." Parasit Vectors 9(1): 396.


(9) The product safety concern in the future
Environmental concern
Leishmania culture requires a certain osmolarity and combination of nutrients. Therefore, our transgenic Leishmania will not survive as soon as it leaves the medium.
Side effects of adjuvant
There are some potential adverse effects underlying every drug and vaccine. More animal test should be carried out and the most efficient dose in human will be selected before clinical trials. In the clinical study, side effect after immunization will be identified and study design will be modified as well. If our product passes the FDA approval, the report of unexpected effect will be always under inspection.


(10) How would your project be used in the real world?
Our Leijuvant can act to facilitate both humeral and cellular immune response which may contribute to the development of vaccine nowadays. Combined with our prediction software, the platform can predict the peptides on MHC molecules and help to optimize the peptide presentation which may benefit to engineer the sequence for further research and developing a more efficient vaccine.