Attributions

Team composition

Team is composed of 11 undergraduate students of biochemistry, biotechnology, medicine and informatics and computer sciences at the University of Ljubljana, two of which finished their undergraduate study during the summer and one of them enrolled in October 2012 to the PhD study of biomedicine.

Team selection

The 2012 Slovenian iGEM team was selected among more than 40 candidates who replied to the team member recruitment announcement posted at the end of December 2011. An initial meeting of candidates with advisers announced the challenge of this year's project - to find a synthetic biological solution for medical therapy using biological drugs. Out of the candidates that attended the initial meeting, 18 candidates submitted their proposal for the project by the end of February.

Eight wet lab team members were selected based on the interviews with team advisers in March while the three computer modeling team members were selected by advisers from the Faculty of informatics and computer sciences. The team started with experimental work towards the end of April and more intensively at the end of June after the summer exams.

Contribution of student team members:

• contributed ideas for the project
• performed all cloning (the ‘masters of cloning’ were mainly Boštjan and Fedja, with help of all the other students, Uroš, Miha, Lucija, Anja, Zala, Urban)
• performed all cell culture experiments (transfection, growth, fluorescence and luminescence reporter and cytokine assays) (Mainly Uroš, Miha, Lucija, Anja and Urban)
• performed all cell encapsulation experiments (Urban)
• preformed all biochemical analysis (WB, fluorescence, luminescence measurements) (Uroš, Miha, Lucija, Anja, Urban)
• modeling (Dušan and Martin (simulations of switches), Maja (pharmacokinetics) in consultation with other team members)
• wrote, distributed and analyzed the results of the survey (Zala and Maja)
• discussed the project and uses of synthetic biology with physicians, patients, experts for ethics and safety, media (all students)

Support of advisers:

• basic training of students for techniques of recombinant DNA and cell biology (Jan, Rok, Vida, Tina, Alja, Anže, Andreja)
• technical support on flow cytometry (Alja, Mojca, Tina)
• technical support with confocal microscopy (Mojca, Tina, Andreja)
• technical support with cell microencapsulation (Anže)
• establishing connections with high school headmasters (Alja, Anže, Rok, Andreja)
• discussing the results and editing the wiki (all advisers)
• soliciting funds for the project (all advisers)

Induction in mammalian cells

At the initiation of the project the host lab only had a tetracycline induction system. Induction systems with ecdysone and rapalogues were purchased (Agilent Technologies and Clontech Laboratories, respectively).

We thank Prof. Martin Fussenegger who kindly provided all the genetic components for establishing the erythromycin and pristinamycin induction systems.

All five induction systems were adapted for the project purposes by the addition of TAL regulators. Because of the MTA limitations the induction systems could not be deposited into the Registry.

TAL effectors

The 2010 Slovenian iGEM team prepared NicTAL, a TAL effector designed to bind to the teto1 operator, which however did not work as expected so the team performed experimental work using zinc finger proteins instead. This year's team discovered that a subdomain of TAL essential for the activity, which was not known at that time, was missing from NicTAL. The missing domain was added and NicTAL activator and repressor constructs were prepared and their functionality was demonstrated.

KRAB domain, VP16 domain and minimal promoter (Pmin) were contributed by the host lab.

TAL effectors 59, 95 and 97 were obtained via AddGene from Joung's lab, "Biobricked" and used for the construction of TAL activators, repressors and genetic switch devices. The team prepared TAL-based activators and repressors by adding VP16 and KRAB domain, respectively, where all wet lab team members participated in the experimental work. Different positions of KRAB with respect to TAL DNA- binding domain and number of KRAB domains were investigated by team members (Lucija).

Gene synthesis

• binding sites for TAL effectors were ordered from IDT
• DNA blocks for the assembly of Pseudoalteromonas elyakovii alginate lyase with an optimized codon sequence where ordered from IDT and cloned by Urban.

Pseudomonas aeruginosa alginate lyase

A P. aeruginosa strain was generously donated by Prof. Guillermo Martínez de Tejada, whom we kindly thank for his support.

Cell microencapsulation

The host lab obtained cell microencapsulator in the middle of 2011.

Team advisor Anže supervised microencapsuation, which was performed by Urban.

Therapeutic effectors

The host lab contributed a clone for IL1RA (anakinra), while all other effectors (IFN-alpha, MICA, VEGF, PDGF) were purchased from Sino Biological and were cloned by team members into the appropriate vectors and deposited into the Registry (Lucija, Boštjan and Fedja). Biological activity of anakinra and IFN-alpha and determination of their concentration were measured by team members (Urban, Lucija, Miha).

Cell killing assay

NK cells were from the ATCC. Cell killing assays were performed by microscopy and flow cytometry by students (Anja and Lucija), with supervision on the microscope and flow cytometer by advisers (Mojca, Alja, Tina).

Modeling of switches and pharmacokinetics

All modeling was performed by student members (Dušan, Martin, Maja in consultation on the biological questions with Boštjan, Miha and Urban).

Wiki

Wiki including all animations and images was prepared by students and edited by the advisers.

3D models

3d models of human anatomy used for visualization purposes were obtained from BodyParts3D database, available at http://dbarchive.biosciencedbc.jp/en/bodyparts3d/desc.html

For real-time pharmacokinetic 3D simulation purposes, the models were converted to OpenGL-friendly format using obj2opengl, available at: http://heikobehrens.net/2009/08/27/obj2opengl

TAL regulators in Xenopus laevis

The experiment with TALA:VP16 and 10x[TALA]_pMIN_mCitrine in Xenopus laevis was performed by team Evry, during our colaboration.