Revision as of 13:05, 1 November 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 the 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 the 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.

The 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.

Characterization of RNA II (Vilnius-Lithuania 2017)

RNA I inactivation in wild type replicon

References