Revision as of 20:43, 1 November 2017

SynORI inducible global copy number control device

This device is a part of SynORI framework. It is a fully functional synthetic origin of replication that sets a induced copy number of every plasmid group in the system. Different concentrations of ROP protein provide a different copy number of plasmids.

Note: introducing this device into a SynORI framework will lower the plasmid copy number of every group in system.

Devices from the same series that have different promoters: part:BBa_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 Anderson).

See how this part fits into the whole SynORI framework by pressing here!

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. That means it can act as a global copy number modulator, which bypasses the selective control of each plasmid group.

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.

Characterization of RNA II (Vilnius-Lithuania 2017)

Characterization of the inducible ROP protein

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.