Revision as of 13:15, 31 October 2017

SynORI constitutive copy number device (Group D-GC, with RNA II)

This device is a fully functional synthetic origin of replication that sets a constitutive copy number to a plasmid. Different concentrations of RNA I gene provide a different copy number of a plasmid. As it is from group D, its RNA I will only bind to RNA II of the same composite part and not other plasmid groups that might be in a cell.

Three main devices were used for the SynORI proof of concept: Part:BBa_K2259078, Part:BBa_K2259079, Part:BBa_K2259080.

Sequence and Features

Introduction

Biology

ColE1 plasmid replication overview

Figure 1. Main principles of ColE1 plasmid family replication. (Citation needed)

ColE1-type plasmid replication begins with synthesis of plasmid encoded RNA II (also called primer transcript) by RNA polymerase which initiates transcription at a site 555bp upstream of origin of replication. The RNA transcript forms a RNA - DNA hybrid with template DNA near the origin of replication. Hybridized RNA is then cleaved at the replication origin by RNAse H and serves as a primer for DNA synthesis by DNA polymerase I (Figure 1. A).

Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I . Synthesis of RNA I starts 445 bp upstream of the replication origin and proceeds in the direction opposite to that of RNA II synthesis, and terminates near the RNA II transcription initiation site. RNA I binds to RNA II and thereby prevents formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).

For RNA I to inhibit primer formation, it must bind before the nascent RNA II transcript extends to the replication origin. Consequently, the concentration of RNA I and the rate of binding of RNA I to RNA II is critical for regulation of primer formation and thus for plasmid replication.

Interaction between RNA I and RNA II can be amplified by Rop protein, see part:BBa_K2259010.

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]!

Specific RNA I and RNA II versions in SynORI framework

As RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this fact to our advantage in order to engineer different plasmid groups by adding unique, group - specific sequences to RNA I and RNA II stem loops.

For example if there are two plasmid groups in a cell - A and B - RNA II of A group
would only interact with RNA I A, and not RNA I B.

The inactivation and transfer of RNA I gene away from RNA II allows us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other.

Since there are three stem loops responsible for RNA I – RNA II interaction for each of the plasmid group we have decided to:

• Use two different unique sequences in the first two stem loops, in order to maximize same group specificity.
• For the third loop, we have decided to keep RNA II unchanged, and add either G/C mutations (GC type RNA I) or make RNA I completely non-complement to RNA II (NC type RNA I).

We did not want to introduce new specific sequences into the third loop of RNA II sequence. That is because according to literature <links> RNA II secondary structures at third loop structure are very sensitive to any mutations and has a high chance of ruining the replication initiation. Just because we chose not to interfere with the third loop of RNA II, we could not leave RNA I gene unchanged. If every group would have the fully compatible third loop, the background cross-group inhibition would be too large.

So now we have 5 different RNA II genes corresponding to groups A B C D and E.

Also, we have 10 different RNA I alternatives: A , B, C, D, E with each having a version of either G/C or NC mutations.

So for example if we have a part named RNA I (B-NC), it means: This RNA will only selectively regulate RNA II molecule by having specific B group sequences in first two stem loops. Also, in the third stem loop every nucleotide is not complementary to RNA II third loop.

These different plasmid groups (A-E) can then be co-maintained in cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.

Figure 1. RNA I AND II group interaction example

Origin of RNA I biobrick

In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in ColE1 origin of replication. That, however, was not a trivial task, as ColE1 ORI is an antisense system, which means that by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation (Find how this problem was solved at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]). This is the main reason why, in SynORI framework, the wildtype ColE1 ORI is split into two different parts - RNR I and RNA II .

Characterization (Vilnius-Lithuania 2017)

SynORI proof of concept

To be updated!

References