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</div> | </div> | ||
<div class="slide-left-content slide-left-content-6"> | <div class="slide-left-content slide-left-content-6"> | ||
+ | <div class="owl-carousel"> | ||
+ | <div class="slide slide-image" style="background-image: url('http://placehold.it/800x600/2f3b4b/ffffff')"></div> | ||
+ | <div class="slide slide-image" style="background-image: url('http://placehold.it/800x600/000000/ffffff')"></div> | ||
+ | <div class="slide slide-image" style="background-image: url('http://placehold.it/800x600/72808d/ffffff')"></div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="slide-left-content slide-left-content-1"> | ||
<div class="owl-carousel"> | <div class="owl-carousel"> | ||
<div class="slide slide-image" style="background-image: url('http://placehold.it/800x600/2f3b4b/ffffff')"></div> | <div class="slide slide-image" style="background-image: url('http://placehold.it/800x600/2f3b4b/ffffff')"></div> | ||
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<div class="right-content"> | <div class="right-content"> | ||
− | + | <div class="slide-right-content slide-right-content-1 is-current"> | |
− | <h1> | + | <h1>Determining the plasmid copy number</h1> |
<div class="category">Design</div> | <div class="category">Design</div> | ||
− | <p> | + | <p>TODO</p> |
<div class="readmore" data-modal="1">read more</div> | <div class="readmore" data-modal="1">read more</div> | ||
</div> | </div> | ||
<div class="slide-right-content slide-right-content-2 hidden"> | <div class="slide-right-content slide-right-content-2 hidden"> | ||
− | <h1> | + | <h1>Plasmid copy number control</h1> |
<div class="category">Design</div> | <div class="category">Design</div> | ||
− | <p> | + | <p>Flexible copy number control is the core of our framework, which is based on re-engineered ColE1 origin of replicon.</p> |
<div class="readmore" data-modal="2">read more</div> | <div class="readmore" data-modal="2">read more</div> | ||
</div> | </div> | ||
<div class="slide-right-content slide-right-content-3 hidden"> | <div class="slide-right-content slide-right-content-3 hidden"> | ||
− | <h1> | + | <h1>Multiple plasmid groups</h1> |
<div class="category">Design</div> | <div class="category">Design</div> | ||
− | <p> | + | <p>Multi-plasmid framework would not be much without multiple plasmids. We have equiped our synthetic origin of replication with specific recognition sequences to create unique plasmid groups.</p> |
<div class="readmore" data-modal="3">read more</div> | <div class="readmore" data-modal="3">read more</div> | ||
</div> | </div> | ||
<div class="slide-right-content slide-right-content-4 hidden"> | <div class="slide-right-content slide-right-content-4 hidden"> | ||
− | <h1> | + | <h1>Global copy number regulation</h1> |
<div class="category">Design</div> | <div class="category">Design</div> | ||
− | <p> | + | <p>A global parameter to control every plasmid group at the same time. Introducing Rop protein!</p> |
<div class="readmore" data-modal="4">read more</div> | <div class="readmore" data-modal="4">read more</div> | ||
</div> | </div> | ||
<div class="slide-right-content slide-right-content-5 hidden"> | <div class="slide-right-content slide-right-content-5 hidden"> | ||
− | <h1> | + | <h1>SynORI selection system</h1> |
<div class="category">Design</div> | <div class="category">Design</div> | ||
− | <p> | + | <p>Having multiple plasmids in a cell means using multiple antibiotics. Does it?</p> |
<div class="readmore" data-modal="5">read more</div> | <div class="readmore" data-modal="5">read more</div> | ||
</div> | </div> | ||
<div class="slide-right-content slide-right-content-6 hidden"> | <div class="slide-right-content slide-right-content-6 hidden"> | ||
− | <h1> | + | <h1>Active partitioning system</h1> |
− | <div class="category"> | + | <div class="category">Design</div> |
− | <p> | + | <p>If at least one of the plasmid group has a low copy number, they require extra care to not be lost at cell division. Therefore, SynORI framework incorporates a <i>special</i> partitioning system derived from pSC101 replicon. </p> |
− | + | ||
<div class="readmore" data-modal="6">read more</div> | <div class="readmore" data-modal="6">read more</div> | ||
</div> | </div> | ||
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<a href="/Team:Vilnius-Lithuania/Design" class="active">Design</a> | <a href="/Team:Vilnius-Lithuania/Design" class="active">Design</a> | ||
<ul> | <ul> | ||
− | + | <li class="is-current"><div class="sli-item sli-item-1">Determining the plasmid copy number</div></li> | |
− | <li><div class="sli-item sli-item- | + | <li><div class="sli-item sli-item-2">Plasmid copy number control</div></li> |
− | <li><div class="sli-item sli-item- | + | <li><div class="sli-item sli-item-3">Multiple plasmid groups</div></li> |
− | <li><div class="sli-item sli-item- | + | <li><div class="sli-item sli-item-4">Global copy number regulation</div></li> |
− | <li><div class="sli-item sli-item- | + | <li><div class="sli-item sli-item-5">SynORI selection system</div></li> |
+ | <li><div class="sli-item sli-item-6">Active partitioning system</div></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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</div> | </div> | ||
− | |||
<div class="modal modal-1"> | <div class="modal modal-1"> | ||
+ | <div class="modal-close"></div> | ||
+ | <div class="modal-content"> | ||
+ | <h1>Determing the plasmid copy number</h1> | ||
+ | <h5>TODO</h5> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="modal modal-2"> | ||
<div class="modal-close"></div> | <div class="modal-close"></div> | ||
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</div> | </div> | ||
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<div class="modal-close"></div> | <div class="modal-close"></div> | ||
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</div> | </div> | ||
− | <div class="modal modal- | + | |
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<h2>Split antibiotic – 2 plasmids system</h2> | <h2>Split antibiotic – 2 plasmids system</h2> | ||
<p>One of the essential parts of synthetic biology are plasmids. However, bacterial plasmid systems require a unique selection, usually an antibiotic resistance gene, to stably maintain the plasmids. As the number of different plasmid groups used in a single cell rise, the need for more selection markers grows. In addition to raising the issue of biosafety, the use of multiple antibiotic resistance genes destabilizes the functionality of the cells. To address this problem a protein granting the resistance to aminoglycoside family antibiotics, called amino 3'-glycosyl phosphotransferase (APH(3')), was split into two subunits by Calvin M. Schmidt et al. </p><p> | <p>One of the essential parts of synthetic biology are plasmids. However, bacterial plasmid systems require a unique selection, usually an antibiotic resistance gene, to stably maintain the plasmids. As the number of different plasmid groups used in a single cell rise, the need for more selection markers grows. In addition to raising the issue of biosafety, the use of multiple antibiotic resistance genes destabilizes the functionality of the cells. To address this problem a protein granting the resistance to aminoglycoside family antibiotics, called amino 3'-glycosyl phosphotransferase (APH(3')), was split into two subunits by Calvin M. Schmidt et al. </p><p> | ||
− | According to the obscure guidelines we split an unmodified neo gene sequence between 59 and 60 amino acid residues. Two subunits were termed | + | According to the obscure guidelines we split an unmodified neo gene sequence between 59 and 60 amino acid residues. Two subunits were termed a-neo and ß-neo. Furthermore, we added additional termination codon at the end of an a-neo fragment for the translation to stop. No other start codons were included into the ß-neo subunit as the gene was designed for toehold switch system. Despite the fact that ß-neo subunit had no start codon, the split antibiotic system worked perfectly when coupled with a standard promoter and a ribosome binding site (BBa_K608002). Consequently, a split antibiotic resistance gene provides a selection system to stably maintain two different plasmids. |
</p><p><div class="img-cont"> | </p><p><div class="img-cont"> | ||
<img src="http://placehold.it/800x450" alt="img"> | <img src="http://placehold.it/800x450" alt="img"> | ||
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</div> | </div> | ||
− | <p>Although the linker sequence adds additional 10 amino acid residues to the peptide, we reasoned that it will not affect the function of split antibiotic. Toehold switches are unlocked when an RNA trigger binds to the 5’ end of the toehold and initiates RNA duplex formation, which releases the locked RBS and reveals linker start codon. We concluded, that if the toehold sequences were added in front of | + | <p>Although the linker sequence adds additional 10 amino acid residues to the peptide, we reasoned that it will not affect the function of split antibiotic. Toehold switches are unlocked when an RNA trigger binds to the 5’ end of the toehold and initiates RNA duplex formation, which releases the locked RBS and reveals linker start codon. We concluded, that if the toehold sequences were added in front of a- and ß-neo gene fragments, the translation would require trigger RNA to initiate protein synthesis.</p><p> |
− | Toeholds and their corresponding triggers design sequences were used as described by A. A. Green et al. with some modifications. First of all, it is important to note, that a “scar” which is made between biobrick prefix for protein coding sequences and suffix, contains a termination codon at the 3’ end. Therefore, it was necessary to use the other form of prefix for | + | Toeholds and their corresponding triggers design sequences were used as described by A. A. Green et al. with some modifications. First of all, it is important to note, that a “scar” which is made between biobrick prefix for protein coding sequences and suffix, contains a termination codon at the 3’ end. Therefore, it was necessary to use the other form of prefix for a- and ß-neo genes, as the translation proceeds from one biobrick to another. Furthermore, seeing that the “scar” produced when joining two biobricks is 8 base pairs, we included an additional T nucleotide at the end of linker sequence to ensure the translation stays in frame to the a- and ß-neo genes. We constructed a system, which includes two toehold riboregulators (termed toehold 1 and toehold 2) upstream of a- and ß-neo genes in two different plasmids. The corresponding activating RNA triggers (name trigger 1 and trigger 2) were placed into additional two plasmids under constant expression. All the parts used together complete a 4-plasmid selection system - two distinct trigger RNAs are expressed by two different plasmids in order to unlock the translation of toehold controlled a- and ß-neo peptides to form a complete amino 3'-glycosyl phosphotransferase. For this reason, if one plasmid is lost, the end product – a/ß dimer APH(3') is not formed, therefore bacteria lose their antibiotic resistance. |
</p> | </p> | ||
<p><div class="img-cont"> | <p><div class="img-cont"> | ||
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</p> | </p> | ||
<h2>Phage control – 5 plasmids system</h2> | <h2>Phage control – 5 plasmids system</h2> | ||
− | <p>The SynOri selection system circuit could be expanded by including additional transcription factor which induced the transcription of previously described RNA triggers. The fifth plasmid would house a transcription factor for the initiation of whole system. Phage modified promoter is perfect for this task, as it is activated by cI lambda peptide and repressed by cI 434 peptide with minimal leakage. Both of the RNA triggers - 1 and 2 - were placed under control of phage modified promoter. Furthermore, downstream of the trigger gene we included cI 434 repressor under constant expression to ensure minimal leakage of the promoter. The fifth plasmid was built to constantly express cI lambda – the activator of phage promoter. In the absence of this plasmid, the gene circuit cannot function, as the trigger RNA transcription is repressed by constant cI 434 expression and toehold switches lock the translation of | + | <p>The SynOri selection system circuit could be expanded by including additional transcription factor which induced the transcription of previously described RNA triggers. The fifth plasmid would house a transcription factor for the initiation of whole system. Phage modified promoter is perfect for this task, as it is activated by cI lambda peptide and repressed by cI 434 peptide with minimal leakage. Both of the RNA triggers - 1 and 2 - were placed under control of phage modified promoter. Furthermore, downstream of the trigger gene we included cI 434 repressor under constant expression to ensure minimal leakage of the promoter. The fifth plasmid was built to constantly express cI lambda – the activator of phage promoter. In the absence of this plasmid, the gene circuit cannot function, as the trigger RNA transcription is repressed by constant cI 434 expression and toehold switches lock the translation of a/ß APH(3'). When the final component of the circuit is present, the cI lambda activator enhances the transcription of both RNA triggers. The transcribed triggers then unlock the translation of a/ß neo peptides which form an active protein and confer the resistance to aminoglycoside family antibiotics. |
</p> | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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