Difference between revisions of "Part:BBa K2259072"
Line 3: Line 3:
 
<partinfo>BBa_K2259072 short</partinfo>
 
<partinfo>BBa_K2259072 short</partinfo>
  
−
RNA II acts as a plasmid replication initiator. The transcript folds into a secondary structure which stabilizes the interaction between the nascent RNA and the plasmids DNA. This RNA-DNA hybrid is attacked by RNase H, which cleaves the RNA strand, exposing a 3' hydroxyl group. This allows the extension of the leading strand by DNA polymerase I and consequently, the start of plasmid replication.
+
This construct is an intermediate to full SynORI global copy number control device. It consists of an Anderson promoter, Anderson RBS, ROP protein and a double terminator.
 +
 
 +
When combined with RNA II ([[part:BBa_K2259068]]) this device lowers plasmid copy number of each plasmid group in the system.  
  
−
*Caution! <B>RNA II (Group A)</b> indicates that this plasmid only interacts with regulatory <B>RNA I (Group A)</b> <LINK TO RNA I A> from SynORI (framework for multi-plasmid systems) collection and is stable when placed with other SynORI plasmid groups. RNA II A will not be regulated with RNA I from another group!
 
  
 
See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
 
See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
Line 35: Line 36:
  
 
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]].
 +
 +
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)==
 
==Usage with SynORI (Framework for multi-plasmid systems)==
Line 42: Line 45:
 
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===
 
−
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.
 
−
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.>
 
−
 
−
===This device in SynORI===
 
−
This is a constitutive global copy number modulator device which lowers plasmid copy number of every group in the system bypassing the selective control of different groups. These constitutive devices can be used with different Anderson promoters to select a different copy number.
 
−
 
−
Devices from the same series that have different Anderson promoters:  [[part:BBa_K2259072]] (0 Anderson), [[part:BBa_K2259073]] (0.15 Anderson), [[part:BBa_K2259074]] (0.24 Anderson).
 
−
 
−
 
−
 
−
See the [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).
 
  
−
===Further details===
+
=Characterization=
−
For more background information and indepth insight on this part's design please see the individual part pages of [[part:BBa_K2259000]] and [[part:BBa_K2259010]].
+
  
−
=Characterization of RNA II (Vilnius-Lithuania 2017)=
+
In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259052]].
−
==RNA I inactivation in wild type replicon==
+
  
 
==References==
 
==References==
 
<references />
 
<references />

Revision as of 22:35, 31 October 2017


Rop protein with anderson (0) promoter for global plasmid copy number control

This construct is an intermediate to full SynORI global copy number control device. It consists of an Anderson promoter, Anderson RBS, ROP protein and a double terminator.

When combined with RNA II (part:BBa_K2259068) this device lowers plasmid copy number of each plasmid group in the system.


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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 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

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.

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

Aboutsynoritry1.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]!


Characterization

In order to characterize this construct, it must be cloned next to RNA II gene. Please see part:BBa_K2259052.

References