Latest revision as of 03:38, 2 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.

Rop devices constructed together with different Anderson promoters: part:BBa_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 Anderson), inducible Rhamnose promoter - part:BBa_K2259076

Characterization of Rop protein (Vilnius-Lithuania 2017)

Rop expression

Figure 3. SDS-PAGE of Rop protein induction. M – Thermo Scientific PageRuler Unstained Low Range Protein Ladder; 1 – E. coli soluble proteins fraction without induction after 7 h of growth; 2-5 – cells induced using 1 mM IPTG – hours above tracks indicate different time of growing after induction.

We have first decided to verify the expression of Rop protein to make sure that our designed Rop gene gives appropriate mRNA which is translated in the cell correctly. Rop gene was then placed under inducible T7 promoter. After two hours of growth, E. coli DH5α cells containing plasmid with Rop gene were induced using 1 mM IPTG. Soluble proteins from the cell lysates were separated by centrifugation and then used for SDS-PAGE. Size of Rop protein is 7,5 kDa, so it can be seen below 10 kDa size standard mark. Figure 3 shows, that Rop protein was induced successfully and its quantity increases by prolonging cell growth. It is found in soluble protein fraction which strongly suggests that Rop protein possibly forms an active spatial structure in vivo and might influence RNA I-RNA II duplex formation.

Constitutive Rop protein effect on plasmid copy number

We have then wanted to see if we can add a constitutive Anderson promoter to Rop gene in order to change the copy numbers of a plasmid group. We have cloned 3 different Anderson promoters next to Rop gene and then moved it next to RNA I. We then moved these 3 intermediate parts (part:BBa_K2259072, part:BBa_K2259073, part:BBa_K2259074) into the minimal SynORI vector (part:BBa_K2259092) next to RNA II (part:BBa_K2259075, part:BBa_K2259053, part:BBa_K2259052). We have then calculated the plasmid copy number.

Figure 4. SynORI constitutive global copy number device with Rop under different strength Anderson promoters.

As seen in in the Figure 4, our Rop protein constructs successfully lower the plasmid copy number. Each Anderson promoter increases Rop concentration and consequently, lowers plasmid copy number.

Inducible Rop protein effect on plasmid copy number

We have also investigated Rop protein with inducible Rhamnose promoter in order to have a viable option of inducible copy number control. We have cloned Rop gene next to Rhamnose promoter and RNA I (BBa_K2259070) and then placed this construct next to RNA II (part:BBa_K2259076) in SynORI minimal vector (part:BBa_K2259092).

Figure 5. SynORI inducible global copy number device with different rhamnose concentrations.

These results show that Rhamnose promoter is too strong for Rop protein expression, because even the leakage of promoter at 0 percent induction leads to copy decrease to average 1 copy per cell. That means that cells can barely survive and if they do, the inhibition level is so high they cannot maintain more than one plasmid.

Despite the high expression level this device can still prove to be useful in the future, for example if characterized with an active partitioning system this construct could become a useful tool for extremely low copy plasmid group generator.

Rop protein as a global regulator

When different groups of SynORI system were created the abilty of corresponding RNA I to inhibit the replication of RNA II were measured by calculating the plasmid copy number with and without RNA I in the system

Figure 6. Different RNA II group copy number with and without RNA I of the same group

As can be seen in Figure 6, RNA I introduction into the system has a significant effect on the plasmid copy number of the specific group, thus we can conclude that RNA I works on corresponding RNA II.

To prove that RNA I works only on the specific RNA II, different groups of SynORI devices were placed in a cell by co-transformation and plasmid copy numbers were calculated. SynORI global copy number control devices (part:BBa_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 Anderson)) were co-transformed together with B-GC SynORI device (BBa_K2259078) and (part:BBa_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 Anderson)) with D-GC SynORI device (BBa_K2259079).

Figure 7.SynORI A device with Rop under different Anderson promoters together with SynORI B-GC device.

Figure 8.SynORI A device with Rop under different Anderson promoters together with SynORI D-GC device.

As can be seen in Figure 7 and Figure 8 RNA II A, RNA II B and RNA II D act as different ORIs and their corresponding RNA I inhibits the replication of SynORI groups specifically.

It can also be concluded that Rop protein placed in a single plasmid lowered the plasmid copy number of both plasmid groups, this proves that Rop works by binding to a kissing-loop complex and is able to bypass the individual control of different groups.

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.