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.