Team:Slovenia

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'''Brief description of our project'''
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This year, the Slovenia iGEM team constists of group of undergraduate students of biology, biochemistry, medicine, biotechnology, microbiology and computer science. The goal of our project is to design a bacterial cell system in which we will be able to control the sequence of steps in a multi-step biosynthetic pathway.We will use our approach for synthesis of an industrially important compound as well as information processing.
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<a href="#naslov"><span id="stopnja3a">summary</span></a><a href="/Team:Slovenia/scheme"><span id="stopnja3">scheme</span></a><a href="#achieve"><span id="stopnja3">achievements</span></a>
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<a href="http://2010.igem.org/Team:Slovenia/scheme"><img src="http://2010.igem.org/wiki/images/b/b6/SLOShemasmall.jpg" alt="Team Slovenia project scheme link"></a>
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<div id="naslov" >summary</div>
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|[[Image:Slovenia_team.png|right|frame|Your team picture]]
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Information encoded by triplet nucleotides of DNA determines the order of incorporation of amino acids into proteins. We envisioned an alternative use of DNA as the scaffold for encoding information. We used DNA sequence as a program consisting of series of blocks of nine or more nucleotides. If we add desired functional proteins fused with DNA-binding domains that bind to those DNA blocks, the functional proteins spontaneously arrange along the DNA in a defined order, depending only on the program DNA sequence. This DNA-guided assembly platform provides particularly powerful tool for engineering biosynthetic pathways. We selected six different zinc fingers and determined their binding to their corresponding DNA target sequences using surface plasmon resonance, mobility shift analysis and &beta;-galactosidase reporter assay. FRET effect was used to demonstrate simultaneous binding of four different zinc fingers with split fluorescent proteins to the program DNA in mammalian cells. The real world application of the DNA-guided biosynthesis was demonstrated on a violacein biosynthetic pathway consisting of five enzymes. In addition to violacein the native biosynthetic pathway produces large amount of a green side product deoxychromoviridans. In our designed biosynthetic pathway, however, this side product was absent. Furthermore, the yield of violacein production was 6-fold higher in the presence of DNA program coding for the linear order of enzymes in comparison to the scrambled DNA program. These results demonstrate that the correct order of enzymes in the biosynthetic assembly is important rather than just enzyme clustering. Artificial DNA-binding domains also have great potential for information processing and our modeling demonstrates the potentials for expanding repertoire and features of genetic oscillators.
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|align="center"|[[Team:Slovenia | Team Example]]
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<!--- The Mission, Experiments --->
 
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In conclusion, our project demonstrates novel application of DNA as the carrier of information in synthetic biology. With over 700 available characterized zinc fingers almost limitless number of combinations is on hand, providing a strong support for maturation of synthetic biology from craft towards the engineering science. <html>
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!align="center"|[[Team:Slovenia|Home]]
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!align="center"|[[Team:Slovenia/Team|Team]]
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!align="center"|[http://igem.org/Team.cgi?year=2010&team_name=Slovenia Official Team Profile]
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!align="center"|[[Team:Slovenia/Project|Project]]
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!align="center"|[[Team:Slovenia/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Slovenia/Modeling|Modeling]]
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!align="center"|[[Team:Slovenia/Notebook|Notebook]]
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!align="center"|[[Team:Slovenia/Safety|Safety]]
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<font size="3" color="#000000"><b>We would like to thank our following sponsors:</b></font>  
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<a href="/Team:Slovenia/ABOUT_US"><img width="484" src="http://2010.igem.org/wiki/images/0/0e/SLOSkupinska.jpg"></a><object style="float:right;" width="409" height="342"><param name="movie" value="http://www.youtube.com/v/MWFV3LKDVyc?fs=1&amp;hl=sl_SI"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/MWFV3LKDVyc?fs=1&amp;hl=sl_SI" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="430" height="342"></embed></object>
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<div id="naslov"  >achievements</div>
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<div id="achieve" >
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<li>We designed a new platform of DNA-guided scaffold to      arrange various functional protein domains in a defined linear order &nbsp;</li>
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<li>We prepared biobricks and characterized binding of six      distinct zinc finger proteins to their target DNA binding sites by various      methods both in vitro (SPR, EMSA, sequence-enabled reassembly of split      fluorescent proteins) and in vivo by &beta;-galactosidase repression assay</li>
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<li>We proved simultaneous binding of 4 zinc finger      proteins to adjacent sites on program DNA by FRET</li>
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<li>We designed chimeric violacein biosynthetic enzymes with      added zinc finger&nbsp;DNA binding domains. In the presence of&nbsp;correct      DNA program the yield of violacein production improves 6 fold. </li>
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<li>We suppressed formation of side reaction product      deoxychromoviridans in the violacein biosynthetic pathway in the presence      of DNA program</li>
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<li>We performed both deterministic and stochastic      simulations of Smolen oscillator and repressilator&nbsp;comprising zinc      finger based repressors using realistic kinetic parameters      and Poisson-distributed time delay. In these models we demonstrated that this system      exhibits oscillations only in topologies comprising odd number of repressor elements while the increased number increases the stability and extends      the time constant of oscillator. </li>
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<li>We demonstrated the functionality of designed DNA      binding domains as building blocks of the genetic oscillator</li>
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<li>We submitted 151&nbsp;parts to the registry, which can      be used for application of our DNA-guided platform for biosynthesis,      building oscillators, characterizing DNA binding domains etc.</li>
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<a href="http://www.ki.si/" target="_blank"><img src="http://2008.igem.org/wiki/images/a/a2/KI.gif" width="200" height="130" /></a><br>
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National Institute of Chemistry Slovenia
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<a href="http://www.fri.uni-lj.si/" target="_blank"><img src="http://2010.igem.org/wiki/images/6/67/Fri_slo.jpg" width="100" height="65" /></a><br>
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Faculty of Computer  <br>and Information Science
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<a href="http://www.ad-futura.si" target="_blank"><img src="http://2008.igem.org/wiki/images/3/38/Adfutura.gif" width="200" height="130" /></a><br>
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Ad futura
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Slovenian Research Agency
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<a href="http://www.osir.si/" target="_blank"><img src="http://2010.igem.org/wiki/images/d/de/Osir.jpeg" width="150" height="104" /></a><br>
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OSIR
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KRKA d.d, Novo mesto
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GENEART
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MrGene
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Kemomed d.o.o.
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Majbert
 
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<a href="http://www.mikro-polo.si/" target="_blank"><img src="http://2010.igem.org/wiki/images/1/12/Mikro_polo.png" width="174" height="34" /></a><br>
 
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Latest revision as of 23:22, 27 October 2010

Team Slovenia project scheme link
summary
Information encoded by triplet nucleotides of DNA determines the order of incorporation of amino acids into proteins. We envisioned an alternative use of DNA as the scaffold for encoding information. We used DNA sequence as a program consisting of series of blocks of nine or more nucleotides. If we add desired functional proteins fused with DNA-binding domains that bind to those DNA blocks, the functional proteins spontaneously arrange along the DNA in a defined order, depending only on the program DNA sequence. This DNA-guided assembly platform provides particularly powerful tool for engineering biosynthetic pathways. We selected six different zinc fingers and determined their binding to their corresponding DNA target sequences using surface plasmon resonance, mobility shift analysis and β-galactosidase reporter assay. FRET effect was used to demonstrate simultaneous binding of four different zinc fingers with split fluorescent proteins to the program DNA in mammalian cells. The real world application of the DNA-guided biosynthesis was demonstrated on a violacein biosynthetic pathway consisting of five enzymes. In addition to violacein the native biosynthetic pathway produces large amount of a green side product deoxychromoviridans. In our designed biosynthetic pathway, however, this side product was absent. Furthermore, the yield of violacein production was 6-fold higher in the presence of DNA program coding for the linear order of enzymes in comparison to the scrambled DNA program. These results demonstrate that the correct order of enzymes in the biosynthetic assembly is important rather than just enzyme clustering. Artificial DNA-binding domains also have great potential for information processing and our modeling demonstrates the potentials for expanding repertoire and features of genetic oscillators. In conclusion, our project demonstrates novel application of DNA as the carrier of information in synthetic biology. With over 700 available characterized zinc fingers almost limitless number of combinations is on hand, providing a strong support for maturation of synthetic biology from craft towards the engineering science.
achievements

  • We designed a new platform of DNA-guided scaffold to arrange various functional protein domains in a defined linear order  
  • We prepared biobricks and characterized binding of six distinct zinc finger proteins to their target DNA binding sites by various methods both in vitro (SPR, EMSA, sequence-enabled reassembly of split fluorescent proteins) and in vivo by β-galactosidase repression assay
  • We proved simultaneous binding of 4 zinc finger proteins to adjacent sites on program DNA by FRET
  • We designed chimeric violacein biosynthetic enzymes with added zinc finger DNA binding domains. In the presence of correct DNA program the yield of violacein production improves 6 fold.
  • We suppressed formation of side reaction product deoxychromoviridans in the violacein biosynthetic pathway in the presence of DNA program
  • We performed both deterministic and stochastic simulations of Smolen oscillator and repressilator comprising zinc finger based repressors using realistic kinetic parameters and Poisson-distributed time delay. In these models we demonstrated that this system exhibits oscillations only in topologies comprising odd number of repressor elements while the increased number increases the stability and extends the time constant of oscillator.
  • We demonstrated the functionality of designed DNA binding domains as building blocks of the genetic oscillator
  • We submitted 151 parts to the registry, which can be used for application of our DNA-guided platform for biosynthesis, building oscillators, characterizing DNA binding domains etc.