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− | <h1>Trumpas title, per dvi eilutes</h1> | + | <h1>Global copy number regulation</h1> |
− | <h5>Izangos teksto stilius. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor | + | <h5>Rop protein |
− | incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco
| + | </h5> |
− | laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit
| + | <p>Now that we have figured out and engineered a way to regulate plasmid copy number in a group - specific fashion the only control element that is missing is a way to control every group at the same time. |
− | esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident.</h5>
| + | </p><p> |
− | <p>Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien | + | Recall, we have used ColE1 replicon as base of our system. And it has given us a perfect hint on how to achieve our current objective. Wild type ColE1 replicon codes a small homodimeric, four-helix bundle protein called Rop (also known as repressor of primer). |
− | quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
| + | </p><div class="img-cont"> |
− | eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
| + | <img src="http://placehold.it/800x450" alt="img"> |
− | Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
| + | <div class="img-label">Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error |
− | pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero. | + | sit voluptatem. |
− | Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate | + | </div> |
− | cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
| + | </div> |
− | finibus sapien, at mollis eros ante id massa.</p> | + | <p>Literature shows that Rop secondary structure specific, rather than sequence specific. What that means is that rop recognises RNA I - RNA II kissing loop complex. |
− | <h2>H2 title</h2> | + | </p> |
− | <p>Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum. | + | div class="img-cont"> |
− | Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus | + | <img src="http://placehold.it/800x450" alt="img"> |
− | ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate | + | <div class="img-label">Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error |
− | facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
| + | sit voluptatem. |
− | nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.</p> | + | </div> |
− | <p>In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam
| + | </div> |
− | luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
| + | <p>When Rop binds to secondary structures, it increases the binding affinity of RNA I and RNA II and consequently - replication inhibition. |
− | sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
| + | </p><p> |
− | luctus.</p>
| + | As our framework approach is based on specific RNA sequence binding, having a Rop protein in our system is equivalent of having a global copy normal regulator. In theory, the protein should bind to every complex despite the specific interactions of each group, because the binding geometry should stay similar in each case. |
| + | |
| + | </p> |
| <div class="img-cont"> | | <div class="img-cont"> |
| <img src="http://placehold.it/800x450" alt="img"> | | <img src="http://placehold.it/800x450" alt="img"> |
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− | <h2>H2 title</h2> | + | |
− | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur non urna pharetra, rutrum nibh vitae, | + | <p>We have designed Rop protein with an anderson promoter and showed that it can reduce the copy number of single plasmid, and multiple plasmids non-specifically. |
− | pellentesque erat. Curabitur fringilla ipsum id sapien imperdiet sagittis. Nulla vestibulum arcu neque, et
| + | </p> |
− | iaculis lorem vulputate interdum. Nunc accumsan velit ligula, ac euismod felis sagittis quis. Aenean varius
| + | |
− | mollis aliquet. Nam sodales malesuada porttitor. In sed magna sed neque hendrerit ultrices.</p>
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− | <p>Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien | + | |
− | quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
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− | eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
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− | Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
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− | pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
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− | Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
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− | Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus
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− | ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
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− | facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
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− | nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.</p>
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− | luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
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− | sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
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− | luctus.</p>
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− | <p>Pellentesque gravida ipsum quam, eu vulputate elit egestas eu. Ut eros elit, ullamcorper sit amet accumsan
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− | eget, mollis pretium libero. Etiam laoreet scelerisque risus, in bibendum velit dapibus at. Sed tempor dolor
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− | semper elit aliquet, et fermentum metus tristique. Nulla vel iaculis elit. Vivamus et turpis finibus,
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− | rhoncus dui mattis, blandit sem. Maecenas in risus vestibulum, luctus felis ut, malesuada dui. Aenean vitae
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− | nunc ex. Nunc vulputate orci nec erat iaculis auctor a nec lacus. Pellentesque non ligula laoreet,
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− | tristique leo.</p>
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| </div> | | </div> |
| </div> | | </div> |
Plasmid copy number control
Flexible copy number control is the core of our framework, which is based on re-engineered ColE1 origin of replication.
Base of SynORI framework - ColE1 replicon
ColE1 plasmid replicon is based on two antisense RNA molecules: RNA I and RNA II.
Transcript of RNA II forms a RNA-DNA duplex and acts as a primer for DNA polymerase and for that reason is often called replication initiator.
During the transcription of RNA II several different secondary structures can form. Part of the structures are susceptible to the binding of RNA I – a shorter antisense version of RNA II. The interaction between RNA I and RNA II start upon formation of kissing-loop pairs between their anti-complementary secondary structures. If the kissing complex persists 3’ end of RNA I starts forming a zipper-like duplex with complementary single strand RNA II region. This results in replication inhibition, because primer cannot be formed anymore, which is why RNA I is often called replication inhibitor.
The main reasons why we have chosen ColE1 as base for SynORI framework was:
- It is a light system consisting of only two regulatory RNA molecules
- It is biochemically and mathematically well characterized
- Kissing-loop complex formation kinetics allows to predict plasmid group compatibility.
Picking the control type
It immediately becomes clear that in order to control the copy number of a plasmid one could simply change RNA I promoter. But there is a reason why it was never done before!
As RNA I and RNA II are two antisense molecules, changes made to sequence will affect both of them. Location of RNA I promoter coincides with the RNA II secondary structures, which are crucial to replication primer formation.
palyginimas RNR 1 ir RNR 2 (pilnos) antriniu strukturu ir parodymas kad RNR1 promotorius yra ant svarbiu RNR 2 strukturu, taip pat pazymeti kuriu butent.
Even if one could somehow manage to change the RNA I promoter to another one without disabling replication initiation, it would still not be a convenient because picking another promoter would require a large pool of resources every time.
For that reason we have decided not to change or modify RNA I promoter inside the wild type ColE1 origin of replication, but rather to disable it completely and place a copy of it next to RNA II.
Disabling the RNA I promoter
The main problem of inactivating RNA I promoter is that precautions must be taken in order not to change critical secondary structures of RNA II.
We have first acquired a priority mutation list from literature which analyses RNA polymerase binding affinity to -10 and -35 promoter structures and its dependence on point mutations, with mutations causing the largest decrease in affinity being in the top of the list.
Then, we compiled a simulative algorithm which compared every possible combination of -10, -35 mutations and then compared them to predicted RNA II secondary structures made by CoFold, a thermodynamics-based RNA secondary structure folding algorithm that takes co-transcriptional folding into account. We have picked replicon mutants prioritizing:
- Mutants that have unchanged RNA II secondary structures.
- Mutants that are highest in mutation priority list (lowest RNA polymerase affinity).
Tailoring the copy number control
Once RNA I promoter is disabled in the ColE1 origin of replication, it can be moved to another plasmid location and used as a separate unit. Also, RNA I promoter can now be changed without damaging the replication initiation.
RNA I, and consequently, the copy number of a plasmid can now be placed under virtually any signal pattern required.
We have first showed this by placing RNA I under a series of constitutive Anderson promoters and an inducible Rhamnose promoter.
We can now flexibly control the copy number of a plasmid! What comes next?
Trumpas title, per dvi eilutes
Izangos teksto stilius. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor
incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco
laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit
esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident.
Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien
quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
finibus sapien, at mollis eros ante id massa.
H2 title
Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum.
Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus
ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.
In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam
luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
luctus.
Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error
sit voluptatem.
H2 title
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur non urna pharetra, rutrum nibh vitae,
pellentesque erat. Curabitur fringilla ipsum id sapien imperdiet sagittis. Nulla vestibulum arcu neque, et
iaculis lorem vulputate interdum. Nunc accumsan velit ligula, ac euismod felis sagittis quis. Aenean varius
mollis aliquet. Nam sodales malesuada porttitor. In sed magna sed neque hendrerit ultrices.
Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien
quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
finibus sapien, at mollis eros ante id massa.
Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum.
Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus
ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.
In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam
luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
luctus.
Pellentesque gravida ipsum quam, eu vulputate elit egestas eu. Ut eros elit, ullamcorper sit amet accumsan
eget, mollis pretium libero. Etiam laoreet scelerisque risus, in bibendum velit dapibus at. Sed tempor dolor
semper elit aliquet, et fermentum metus tristique. Nulla vel iaculis elit. Vivamus et turpis finibus,
rhoncus dui mattis, blandit sem. Maecenas in risus vestibulum, luctus felis ut, malesuada dui. Aenean vitae
nunc ex. Nunc vulputate orci nec erat iaculis auctor a nec lacus. Pellentesque non ligula laoreet,
sollicitudin diam sit amet, dignissim mi. Duis dictum risus nulla, eu interdum nisl laoreet ut. Integer eu
leo iaculis, finibus ante eget, consequat orci. Donec semper leo et posuere vulputate. Aliquam ut nisl
porta, sagittis tortor accumsan, pellentesque odio. Ut dolor sapien, porttitor vel ex vitae, condimentum
tristique leo.