Revision as of 19:51, 1 November 2017

Rop protein - global copy number inhibitor (SynORI framework)

Rop (also known as a repressor of primer) is a small protein responsible for keeping the copy number of ColE1 and related bacterial plasmids low in E. coli. ROP inhibits plasmid replication of all SynORI framework plasmid groups and other ColE1 replicon plasmids non-specifically.

In SynORI multi-plasmid framework, Rop acts as a global copy number regulator, by reducing the copy number of all the plasmid groups at the same time. It works not by acting specifically to each plasmid group's RNA II and RNA I sequences, but rather binding to kissing-loop complex that mentioned RNA molecules form.

Sequence and Features

Figure 1. Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)

Introduction

Biology

Repressor of primer (ROP) is a small (7.5 kDa) dimeric protein that participates in ColE1 plasmid family copy number control, by increasing affinity between two complementary RNAs - RNA I (Replication inhibitor) and RNA II (Replication activator) (Fig. 1). ^[1] By increasing affinity of the two RNA molecules, Rop decreases the rate of plasmid replication initiation events.

Figure 2. Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.^[2]

Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).

Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

SynORI is a framework for multi-plasmid systems created by Vilnius-Lithuania 2017 which enables quick and easy workflow with multiple plasmids, while also allowing to freely pick and modulate copy number for every unique plasmid group! Read more about [http://2017.igem.org/Team:Vilnius-Lithuania SynORI here]!

Rop protein in SynORI

Rop protein does not recognize specific sequences of RNA I and RNA II molecules but instead recognizes the RNA I - RNA II kissing loop complex secondary structures and stabilizes them. More stable RNA I - RNA II complex leads to lower plasmid copy number, as RNAI binding inhibits replication. That means it can act as a global copy number modulator, which bypasses the selective control of each plasmid group. It is enough to insert the protein in only one group of plasmids, which minimizes the steps needed to assemble a custom SynORI system.

For example, : You have a two-plasmid system, with specific RNA I concentrations set so that first
plasmid group has an average copy number of 100, and another group at 50 copies. Rop protein can be used to 
globally lower the copy number of each group  - from 100 to 50 and  from 50 to 25 copies respectively. 
The degree of copy number reduction depends on Rop concentration in a cell.

It is recommended to use weak RBS and promoter, as low concentration of protein can reduce plasmid copy number significantly.

Characterization of Rop protein (Vilnius-Lithuania 2017)

Constitutive Rop protein effect on plasmid copy number

To be updated!

Inducible Rop protein effect on plasmid copy number

To be updated!

References

1. ↑ Castagnoli L, Scarpa M, Kokkinidis M, Banner DW, Tsernoglou D, Cesareni G. Genetic and structural analysis of the ColE1 Rop (Rom) protein. The EMBO Journal. 1989;8(2):621-629.
2. ↑ Banner DW, Kokkinidis M, Tsernoglou D. Structure of the ColE1 Rop protein at 1.7 Å resolution. J Mol Biol. 1987 m.;196(3):657–75.