Team:Vilnius-Lithuania/Design
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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?
