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<h1>Multiple plasmid groups | <h1>Multiple plasmid groups | ||
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− | <h5> | + | <h5>Multi-plasmid framework would not be much without multiple plasmids. We have equipped our synthetic origin of replication with specific sequences to create unique plasmid groups.</h5> |
− | </h5> | + | <p>PAs RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this feature to our advantage in order to engineer different plasmid groups by adding unique, group-specific sequences to RNA I and RNA II stem loops. |
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</p><p> | </p><p> | ||
− | + | The specific sequences were acquired from Grabow et al., where they have screened a large number of different RNA kissing stem loop complex combinations. They have derived a table of different loop sequences that only bind with each other but do not have any cross interaction to the following loop sequences in the list. | |
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</p><p> | </p><p> | ||
− | The inactivation | + | The inactivation and transfer of RNA I gene away from RNA II allows us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other. |
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</p><div class="img-cont"> | </p><div class="img-cont"> | ||
<img src="http://placehold.it/800x450" alt="img"> | <img src="http://placehold.it/800x450" alt="img"> | ||
− | <div class="img-label"> | + | <div class="img-label"> |
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</div></div> | </div></div> | ||
− | <p>Since there are three stem loops | + | <p>Since there are three stem loops responsible for RNA I-RNA II interaction for each of the plasmid group we have decided to:</p> |
− | </p> | + | |
<ol> | <ol> | ||
− | <li>Use two different unique sequences in the first two | + | <li>Use two different unique sequences in the first two RNR I and RNR II stem loops, in order to maximize same group specificity.</li> |
− | </li> | + | <li> Keep RNA II unchanged for the third loop but change it in the RNR I by adding either G/C mutations to RNA I (GC type RNA I) or making RNA I completely non-complement to RNA II (NC type RNA I).</li> |
− | <li> Keep the third loop | + | |
− | </li> | + | |
</ol> | </ol> | ||
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</p> | </p> | ||
− | <p>According to literature | + | <p>According to literature <links> RNA II secondary structures at third loop are very sensitive to any mutations and has a high chance of ruining the replication initiation. For this reason we did not want to introduce new specific alterations into the third loop of RNA II sequence. Just because we chose not to interfere with the third loop of RNA II, we could not leave RNA I gene unchanged. If every group would have the fully compatible third loop, the background cross-group inhibition would be too large. |
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</p> | </p> | ||
− | <p> | + | <p>So now we have 5 different RNA II genes corresponding to groups A, B, C, D and E. |
− | </p> | + | </p><p> |
− | + | Also, we have 10 different RNA I alternatives: | |
− | + | A , B, C, D, E with each having a version of either G/C or NC mutations.</p> | |
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− | Also, we have 10 different RNA I alternatives: A, B, C, D, E with each having a version of either G/C or NC mutations | + | |
<p>These different plasmid groups can then be co-maintained in cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.</p> | <p>These different plasmid groups can then be co-maintained in cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.</p> | ||
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<div class="img-cont"> | <div class="img-cont"> | ||
<img src="http://placehold.it/800x450" alt="img"> | <img src="http://placehold.it/800x450" alt="img"> | ||
− | <div class="img-label"> | + | <div class="img-label">Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error |
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</div> | </div> | ||
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<p>The plasmid loss rates were measured using a method described by S. M. Deane et al. We wanted to compare plasmid loss rates with and without active partitioning system (PAR) in 2 different vectors. pSB4A5 vector and Base vector 2.0 (BBa_K225908) which we have built from Minimal base vector (BBa_K2259092) were used for the following experiments in E. coli DH5α strain . | <p>The plasmid loss rates were measured using a method described by S. M. Deane et al. We wanted to compare plasmid loss rates with and without active partitioning system (PAR) in 2 different vectors. pSB4A5 vector and Base vector 2.0 (BBa_K225908) which we have built from Minimal base vector (BBa_K2259092) were used for the following experiments in E. coli DH5α strain . | ||
<p></p> | <p></p> | ||
− | <h2>pSB4A5 + mRFP (BBa_J04450)</h2>< | + | <h2>pSB4A5 + mRFP (BBa_J04450)</h2> |
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+ | <div class="img-cont"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/9/90/Justo_psb4a5_%281%29.png" alt="img"> | ||
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+ | </div> | ||
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<div class="img-cont"> | <div class="img-cont"> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | <h2>Base vector 2.0</h2>< | + | <h2>Base vector 2.0</h2> |
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+ | <div class="img-cont"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/a/a6/Pbv_with_and_without_par.png" alt="img"> | ||
+ | |||
+ | </div> | ||
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Figure 3. Results from the second experiment where plasmid loss was evaluated using our base vector 2.0 (Minimal base vector with (Part:BBa_K2259088). Plasmid-containing bacteria decreased significantly in a vector without an active partitioning system, plasmid loss was already visible in a 60th generation. Where vector with an active partitioning system plasmid loss was not observed in a 60th generation, the loss of both vectors increased later on.</p> | Figure 3. Results from the second experiment where plasmid loss was evaluated using our base vector 2.0 (Minimal base vector with (Part:BBa_K2259088). Plasmid-containing bacteria decreased significantly in a vector without an active partitioning system, plasmid loss was already visible in a 60th generation. Where vector with an active partitioning system plasmid loss was not observed in a 60th generation, the loss of both vectors increased later on.</p> | ||
<p> | <p> |
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