Difference between revisions of "Part:BBa K2259065"
 
(One intermediate revision by the same user not shown)
Line 25: Line 25:
 
===ColE1 plasmid replication overview===
 
===ColE1 plasmid replication overview===
  
−
[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]]
+
[[Image:Cole1 horizontal cropped.png|300px| center|]]
−
<b>ColE1-type plasmid replication begins with the synthesis of plasmid encoded RNA II</b> (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).
+
<b><center>Figure 1. Main principles of ColE1 plasmid family replication</center>
  
−
<b>Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I </b>. 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. <b>RNA I binds to RNA II</b> 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.
+
ColE1-type plasmid replication begins with the synthesis of plasmid encoded RNA II</b> (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).<ref>Itoh, T. and Tomizawa, J. (1980). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proceedings of the National Academy of Sciences, 77(5), pp.2450-2454.</ref>
 +
 
 +
<b>Initiation of replication can be inhibited by plasmid encoded small RNA, called RNA I </b>. 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. <b>RNA I binds to RNA II</b> 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).<ref>Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.</ref>
 +
 
 +
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. <ref>Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.</ref>
  
 
The interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
 
The interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
Line 40: Line 43:
 
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]!
 
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]!
  
−
===Regulative RNA II molecule in SynORI===
+
===This device in SynORI===
−
RNA II gene is foundational and central biobrick of SynORI system and by far the only one that is mandatory for a framework to run.
+
When used together with replication initiator - RNA II ([[part:BBa_K2259000]]), this device can form a system with a range of plasmid copy numbers to choose from, as they are reached by different levels of induction.
−
The two main functions of RNA II is as follows:
+
−
# Initiating plasmid replication
+
−
# Interacting with RNA I of specific plasmid group [[#Specific RNA II versions in multi-plasmid systems|(See below)]]
+
−
 
+
−
 
+
−
=== 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.
+
−
 
+
−
<b>For example</b> 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.
+
−
 
+
−
[[Image:RnainteractionIII.png|center|500px|thumb|<b>Figure 1. </b> RNA I AND II group interaction example]]
+
−
 
+
−
See the [https://parts.igem.org/Part:BBa_K2259000:Design 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 the 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 the SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.
+
−
 
+
−
<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
+
  
 
=Characterization=
 
=Characterization=
  
−
In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259068]].
+
In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259091]].
−
 
+
  
 
==References==
 
==References==
 
<references />
 
<references />

Latest revision as of 17:31, 1 November 2017


SynORI Inducible plasmid copy number device

This construct is an intermediate to full SynORI inducible copy number device. It consists of an Rhamnose promoter and ColE1 RNA I gene (without promoter). It can be combined with part:BBa_K2259000 replication initiation part.

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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Introduction

Biology

ColE1 plasmid replication overview

Cole1 horizontal cropped.png
Figure 1. Main principles of ColE1 plasmid family replication


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).[1]

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).[2]

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. [3]

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

Groupspec.png

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]!

This device in SynORI

When used together with replication initiator - RNA II (part:BBa_K2259000), this device can form a system with a range of plasmid copy numbers to choose from, as they are reached by different levels of induction.

Characterization

In order to characterize this construct, it must be cloned next to RNA II gene. Please see part:BBa_K2259091.

References

  1. ↑ Itoh, T. and Tomizawa, J. (1980). Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proceedings of the National Academy of Sciences, 77(5), pp.2450-2454.
  2. ↑ Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.
  3. ↑ Tomizawa, J. (1984). Control of cole 1 plasmid replication: The process of binding of RNA I to the primer transcript. Cell, 38(3), pp.861-870.