Difference between revisions of "Part:BBa K2259053"
Line 3: Line 3:
 
<partinfo>BBa_K2259053 short</partinfo>
 
<partinfo>BBa_K2259053 short</partinfo>
  
−
RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilizes the interaction between the nascent RNA and the origin's DNA. This 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. Lagging strand synthesis is later initiated by a primase encoded by the host cell.
+
This device is a fully functional synthetic origin of replication that sets a constitutive copy number of every plasmid group in the system. Different concentrations of ROP protein provide a different copy number of a plasmids.
 +
 
 +
Note: introducing this device into a SynORI framework will lower the plasmid copy number of every group in system.  
 +
 
 +
Devices from the same series that have different Anderson promoters:  [[part:BBa_K2259052]] (0 Anderson), [[part:BBa_K2259053]] (0.15 Anderson), [[part:BBa_K2259075]] (0.24 Anderson).
 +
 
 +
See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
 +
 
  
  
Line 32: Line 39:
  
 
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 41: Line 46:
 
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 the framework to run.  
+
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.
−
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)]]
+
−
 
+
−
 
+
−
===Specific RNA II versions in multi-plasmid systems===
+
−
 
+
−
RNA II interacts with inhibitory RNA I with three secondary 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
+
Devices from the same series that have different Anderson promoters: [[part:BBa_K2259052]] (0 Anderson), [[part:BBa_K2259053]] (0.15 Anderson), [[part:BBa_K2259075]] (0.24 Anderson).
−
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]]
 
  
−
===Origin of RNA II biobrick===
 
−
In order to flexibly control the synthesis of RNA I (Why RNA I ? <link to RNA I biobrick>), the RNA I gene first needed to be inactivated in the 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 out more about how team Vilnius-Lithuania solved this problem by pressing this link! <LINK REQUIRED>). 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.>
+
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===
 +
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)=
 
=Characterization of RNA II (Vilnius-Lithuania 2017)=

Revision as of 22:13, 31 October 2017


SynORI global copy number control device (Anderson 0.15)

This device is a fully functional synthetic origin of replication that sets a constitutive copy number of every plasmid group in the system. Different concentrations of ROP protein provide a different copy number of a plasmids.

Note: introducing this device into a SynORI framework will lower the plasmid copy number of every group in system.

Devices from the same series that have different Anderson promoters: part:BBa_K2259052 (0 Anderson), part:BBa_K2259053 (0.15 Anderson), part:BBa_K2259075 (0.24 Anderson).

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 685
    Illegal NheI site found at 708
  • 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.

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]!

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_K2259052 (0 Anderson), part:BBa_K2259053 (0.15 Anderson), part:BBa_K2259075 (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

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)

Constitutive Rop protein effect on plasmid copy number

To be updated!

References