Team:UNICAMP-EMSE Brazil/Results/Judging

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JUDGING

Project Abstract:

“Stress and autoimmune diseases cause imbalances in immune system, observed in the biased naive T-CD4+ Lymphocytes differentiation towards T-lymphocyte-‘helper’-1 in autoimmune diseases, or Th2 in stressful condition, favoring cellular or humoral adaptive responses. This can lead to bacterial evasion of host defense system and susceptibility to some pathogens. Since nowadays we are exposed to continuously stress, perhaps we can avoid the negative effects caused by this condition. Thus, a mechanism that counteracts this imbalance is highly desirable. The ability of some bacteria to sense stress hormones such as Catecholamines will be used to produce Interleukin-12 and inducing Th1 lineage. On the other hand, the ability to sense Nitric-Oxide release in inflammatory conditions will be used to trigger Interleukin-10 production, counteracting excessive immunity. A switch control system will sustain the balance. The ‘Jedi Bacteria’ containing these devices would be a useful probiotic to fight against the battle imposed by stress”.

iGEM Medals for Non-Software Teams

  • We believe our team deserves the following medal: Gold

Because we met the following criteria:

  • Requirements for a Bronze Medal:
  • Register the team, have a great summer, and plan to have fun at the Regional Jamboree. YES
  • Successfully complete and submit this iGEM 2011 Judging form. YES
  • Create and share a Description of the team’s project using the iGEM wiki and the team’s parts using the Registry of :*Standard Biological Parts. YES
  • Plan to present a poster and Talk at the iGEM Jamboree. YES
  • Enter information detailing at least one new standard BioBrick Part or Device in the Registry of Standard Biological :*Parts. Including: YES
  • Primary nucleic acid sequence YES
  • Description of function YES
  • Authorship YES
  • Safety notes, if relevant YES
  • Acknowedgment of sources and references YES
  • submit DNA for at least one new BioBrick Part or Device to the Registry.YES
  • Additional Requirements for a Silver Medal:
  • Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device.YES
  • Enter this information and other documentation on the part’s ‘Main Page’ section of the Registry. Part Number (s): BBa_K554013, BBa_K554012 YES
  • Additional Requirements for a Gold Medal: (one OR more)



UNICAMP-EMSE Brazil Parts Design: why are they different to Stanford 2009 parts?

In the section below we aim to explain in details how our parts differ from Stanford 2009's, and why we decided to create novel parts to achieve similar objectives.

Secretion System Changes

Analyzing the pre-existing secretion system from Stanford Team 2009 we have decided to make several changes in order to make it more efficient and to prove that it actually works.

  1. Change the RBS part and its disposition in the transcriptional unit.
    1. Attempting to create a new secretion system, we began by choosing a different but still highly efficient RBS part. And according to the experience made upon RBS deposited parts (BBa_B0034), it was suggested that the RBS chosen by our team (B0030) is as efficient as the one used by Stanford 2009 team (B0034).
    2. To improve translation efficiency, gene sequence fidelity (avoid errors or mistranscription during PCR) and save time during the devices assembling UNICAMP-EMSE team synthesized each coding sequence already fusioned with RBS sequence. On the other hand Stanford 2009 Team did not use translational units.
2. Biobrick Standard Compatibility
  • The secretion system developed by Stanford Team 2009 was not compatible with the systems 10, 12, 21, 23 nor 25. UNICAMP-EMSE Team has removed internal illegal restriction sites presented in the parts Bba_K223055 and BBa_K223057 by codon usage of these sites prior to synthesis. Therefore our team suited the secretion system to be compatible with 10, 12 and 23 standards.
3. Exclusion of non-essential genes
  • In our system we have reduced the number of genes to use only the essential ones, considering that “the simpler, the better”. Evidence (Fath et al, 1993) shows that only TolC, HlyB and HlyD are essential for the system to work, hence we have excluded the gene HlyC, used by Stanford 2009 Team (Part Bba_K223056). Hemolysin C (HlyC), which encodes a 170 aminoacids protein, has no secretion function but facilitates the activation of Hemolysin A protein which can act as an cytotoxic agent for many host cells.
4. HlyB and HlyD
5. Experience

Response System Improvements

UNICAMP-EMSE Team aimed the development of a bacteria to be used as a probiotic and capable of sensing and interfering with stress-caused immune imbalances and related diseases. Instead of using retinoic acid and its pathway genes to act as an immune-suppressor of Th1 overactivation (as used by Stanford 2009 Team) we have chosen to use Interleukin 10 (IL-10). This choice was made upon scientific background describing this molecule as able to act in two important directions we considered interesting to create a immune balance: IL-10 is efficient to establish an Immunosuppression of Th1 lineage (by directly inhibiting IL-2 production by these cells (de Vries, 1995)), inhibit the production of inflammatory signals (de Vries, 1995; Sabat et al, 2010) and also to redirect the immune cells differentiation toward Th2 (Elenkov et al, 1999). The special physiological relevance of this cytokine lies in the prevention and limitation of over-whelming specific and unspecific immune reactions and, in consequence, of tissue damage (Sabat et al, 2010). Additionally, it was observed that IL-10 was efficient in the treatment of chronic gut diseases associated with autoimmunity (Steidler et al., 2000) and also a bacteria, Lactococcus lactis, efficiently secreted biologically active murine IL-10 (Schotte et al., 2000). These data, linked to new delivery technologies (Huyghebaert et al 2005) indicate that IL-10 is suitable to be used as a probiotic.

The mRNA sequence of human IL10 had its expression in E. coli optimized by codon usage and suited to standard 23 through remotion of internal restriction sites. Additionally, RBS sequence was fusioned with the coding sequence. All these modifications were performed prior to sequence synthesis, which enabled us to create the part BBa_K554004.

References

Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000 Aug 25;289(5483):1352-5.

Schotte L, Steidler L, Vandekerckhove J, Remaut E. Secretion of biologically active murine interleukin-10 by Lactococcus lactis.Enzyme Microb Technol. 2000 Dec;27(10):761-765.

Huyghebaert N, Vermeire A, Neirynck S, Steidler L, Remaut E, Remon JP. Secretion of biologically active murine interleukin-10 by Lactococcus lactis. Enzyme and Microbial Technology 27 (2000) 761–765

de Vries JE. Immunosuppressive and anti-inflammatory properties of interleukin 10.Ann Med. 1995 Oct;27(5):537-41.

Sabat R, Grütz G, Warszawska K, Kirsch S, Witte E, Wolk K, Geginat J. Biology of interleukin-10. Cytokine Growth Factor Rev. 2010 Oct;21(5):331-44. Epub 2010 Nov 5.

Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995-1017.

iGEM Prizes

We believe our team deserves the following prizes:

Best Human Practices Advance

  • Our Human Practices Activites were composed by three activities: The DNA Workshop and two lectures given in Brazil by the Brazilian students (CAEB presentation) and in France by the French students (EMSE presentation). The DNA Workshop is a self-propagative educational method we developed, composed by a Genetic Engineering video class and a dynamic Workshop in which the students were stimulated to solve problems by creating a genetic system with the available parts. We believe this method can be used to disseminate knowledge about genetic engineering and synthetic biology in schools. We could test this method and prove its efficacy in providing to the students an overview of these scientific fields and also to make them excited, curious and interested about this area. (See more details here: https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Human_Practices/DNA_workshop). For us, the DNA workshop meant much more than just a judging requirement. We learned and grew with the developed activities. We believe we were able to target many important themes, which we describe completely at https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Human_Practices#General_Results. Additionally, our team selection was a human practice itself. Unlike most teams, half of our students had no previous experience in molecular or synthetic biology and were trained during the project. Four months later we were proud to see them back in France giving a lecture about iGEM and all the acknowledge they acquired. Our Human Practice experience was extremely rich, not only because it was performed in 2 different countries, but also because of the dedication shown by our team students as well as by the students attending the activities, which got deeply involved in the theme. For us, this is the real meaning of a human practice: being an experience that goes beyond our country, beyond our team, beyond 2011.

Best Wiki page

We have built a very complete, interesting, and creative wiki page. Our wiki presents detailed description of the project, methods, results, parts, team, notebook, human practices, safety, profile and sponsors with a lot of didactics and sense of humor. Our parts were posted completely, the registry is really complete and filled with usefull information, and we have modeled their structure through Jmol app.

Best Health or Medicine Project

Our project has a great importance and impacts in general health condition, because is focused on healing immune imbalances caused by stress, which is considered a worldwide major health issue. Stress Wars aims to develop a valuable and useful genetic tool, that can be effectively applied in multiple imbalances situations, not only stress, as auto-immune disease, cancer and vaccine modulation. Considering the impact of our idea, all the knowledge acquired and transmitted, as well as the achievements we’ve gotten along the course of the project, we believe we deserve the gold medal and also the best Health Project award!!!

Best Poster and Presentation

Just wait and see! Considering the quality of our wiki, our didactics and sense of humor you can expect a high level presentation on Jamboree.

Team_Parts

  • Part Number(s):
  • iGEM Safety
  • Attribution and Contributions


References

Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995-1017. Review. Link to PubMed
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