Proof of concept

Controlled plasmid copy number

For the proof of concept of our system, we displayed that plasmid copy number control can be controlled as desired by employing any kind of promoter upstream of the RNA I gene. By mutating the ColE1 origin of replication we liberated the RNA I gene from the origin site. As a consequence of that, we were able to put the RNA I construct under a constant expression of different anderson promoters. The copy number of each plasmid controlled by RNAI expressed by different anderson promoter has been evaluated (BBa_K2259067; BBa_K2259068; BBa_K2259069; BBa_K2259071).

Figure 1. The plasmid copy number of differently expressed RNA I constructs.

As displayed by the results in the fig. 1 we have constructed a particularly expressing RNA I devices that influence the plasmid copy number. By increasing the promoters strength, the concentration of RNA I molecules increases and, as a result of that, plasmid replication is inhibited. We have showed that plasmid copy number is inversely proportional to the strength of promoter that expresses the RNA I molecule.

ColE1 based independent origins of replication and global copy number control.

Our team has designed synthetic origins of replications based on ColE1 replicon. We have demonstrated that two origins of replication do not cross-interact and from independent plasmid groups. By evaluating the plasmid copy number decrease when group A RNA II is present with group B RNA I and vice versa, we have concluded that replicons A and B do not interact with each other.

Figure 2. Cross-interaction assessment of two origins of replication

Next, we have co-transformed both A and B plasmid groups with their corresponding RNA I to determinate the plasmid copy number of each plasmid. Additionally, we have included different levels of ROP protein to assess the global plasmid number control.

Figure 3. Plasmid copy number of two co-transformed groups of plasmids.

As seen from the Fig. 3, we have proved, that both plasmids groups can be evaluated separately from the same sample and, in contrast to A and D group results, the A and B group replicons do not cross-interact because the plasmid copy number stays higher, as compared to interacting A and D replicons. This proves that both replicons function independently because the copy number does not drop, when the plasmids are co-transformed together.

Furthermore, by expressing the protein with different Anderson promoters we have demonstrated that our global copy number control devices work as intendent and lower the copy number of both plasmid groups equally. As seen from the fig. 3, if the ROP protein is expressed, the copy number of both plasmid groups in the distinct experiments react in the same way – by decreasing it for both plasmid groups. This concludes the function of ROP protein as a global plasmid copy number control device.