Revision as of 00:06, 31 October 2017

ColE1 replicon (Deactivated RNA I, placed next to RNA II)

This is a ColE1 replicon with knocked-out RNA I promoter. A copy of RNA I is then placed next to RNA II.

This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely, because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.

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

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.

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]!

Purpose of this construct in SynORI framework development

This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely, because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.

RNA II and RNA I in the engineering of unique plasmid groups for multi-plasmid system

RNA II molecule interacts with inhibitory RNA I molecule with three secondary structure RNA stem loops. In order to create plasmid groups with independent copy number control, one group's RNA II molecule must interact only with the same group's RNA I molecule.

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.

Figure 1. RNA I AND II group interaction example

See the Design section or [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).

Origin of RNA II biobrick

If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?

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, because by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation. This is the main reason why, in SynORI framework, the wildtype ColE1 ORI is split into two different parts - RNR I and RNA II .

<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>

Characterization of RNA II (Vilnius-Lithuania 2017)

RNA I inactivation in wild type replicon

References