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.

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?

Multiple plasmid groups

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.

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.

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.

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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Since there are three stem loops responsible for RNA I–RNA II interaction for each of the plasmid group we have decided to:

1. Use two different unique sequences in the first two RNR I and RNR II stem loops, in order to maximize same group specificity.
2. 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).

According to literature 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.

So now we have 5 different RNA II genes corresponding to groups A, B, C, D and E.

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.

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.

Global copy number regulation

Rop protein

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.

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).

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.

When Rop binds to secondary structures, it increases the binding affinity of RNA I and RNA II and consequently - replication inhibition.

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.

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.

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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.

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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.

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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.

Active partitioning system

SynORI framework gives the opportunity to have low copy plasmid groups, yet in order for them not to be lost during cell division, there must be a mechanism that actively keeps plasmids in the cell.

We have looked into different active partitioning systems and first chose to characterize and apply a Staphylococcus aureus type II plasmid segregation system to our framework. Yet, after a lot of consideration we have decided to search for alternatives. The main reason was that partitioning system of S. aureus is rather large, almost 49 kDa, as it codes two large proteins for segregation.

We have then stumbled upon a described pSC101 plasmid stability region which is a lot different from its counterpart. It does not seem to encode any protein but rather contains a binding site for DNA gyrase (Wahle and Kornberg, 1988). Most importantly, the regulatory region is only 400 base pairs long.

It has been showed that pSC101 plasmids with partial deletions of stability region have decreased supercoiling and are extremely unstable. This has led to the proposal that gyrase-generated negative supercoiling establishes a DNA conformation which enables plasmids to interact with E. coli structures and distribute them to daughter cells during cell division (Miller et al., 1990)

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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.

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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.

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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.