Difference between revisions of "Part:BBa K2259076"

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===About SynORI===
 
===About SynORI===
 
[[Image:Aboutsynoritry1.png|600px|center|]]
 
[[Image:Aboutsynoritry1.png|600px|center|]]
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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]!
  
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===Regulative RNA II molecule in SynORI===
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===Rop protein in SynORI===
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RNA II gene is foundational and central biobrick of SynORI system, and by far the only one that is mandatory for framework to run.
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Rop protein does not recognise specific sequences of RNA I and RNA II molecules, but instead recognises the RNA I - RNA II kissing loop complex secondary structures. That means it can act as a <b>global copy number modulator</b>, which bypasses the selective control of each plasmid group.
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The two main functions of RNA II is as follows:
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  <b>For example:</b> You have a ''two-plasmid system'', with specific RNA I concentrations set so that
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# Initiating plasmid replication
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  first plasmid group has an average copy number of ''100'', and another group at ''50'' copies. Rop
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# Interacting with RNA I of specific plasmid group [[#Specific RNA II versions in multi-plasmid systems|(See below)]]
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protein can be used to <b>globally lower the copy number of each group </b> - from 100 to 50 and
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from 50 to 25 copies respectively. The degree of copy number reduction depends
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on Rop concentration in a cell.
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=== RNA II and RNA I in the engineering of unique plasmid  groups for multi-plasmid system===
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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.
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  <b>For example</b> if there are two plasmid groups in a cell - A and B - RNA II of A group
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  would only interact with RNA I A, and not RNA I B.
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[[Image:RnainteractionIII.png|center|500px|thumb|<b>Figure 1. </b> RNA I AND II group interaction example]]
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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).
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=Characterization of RNA II (Vilnius-Lithuania 2017)=
 
=Characterization of RNA II (Vilnius-Lithuania 2017)=

Revision as of 23:48, 30 October 2017


SynORI inducible global copy number control device

This device is part of SynORI framework. It is used to induce a certain copy number of a plasmid.

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]



Figure 1. Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)

Introduction

Biology

Repressor of primer (ROP) is a small dimeric protein that participates in ColE1 plasmid family copy number control, by increasing affinity between two complementary RNAs - RNA I (Replication inhibitor) and RNA II (Replication activator) (Fig. 1). [1] By increasing affinity of the two RNA molecules Rop decreases the rate of of plasmid replication initiation events.

Figure 2. Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.[2]

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]!

Rop protein in SynORI

Rop protein does not recognise specific sequences of RNA I and RNA II molecules, but instead recognises the RNA I - RNA II kissing loop complex secondary structures. That means it can act as a global copy number modulator, which bypasses the selective control of each plasmid group. 

For example: You have a two-plasmid system, with specific RNA I concentrations set so that
first plasmid group has an average copy number of 100, and another group at 50 copies. Rop 
protein can be used to globally lower the copy number of each group  - from 100 to 50 and 
from 50 to 25 copies respectively. The degree of copy number reduction depends
on Rop concentration in a cell.

Characterization of RNA II (Vilnius-Lithuania 2017)

RNA I inactivation in wild type replicon

References

  1. ↑ Castagnoli L, Scarpa M, Kokkinidis M, Banner DW, Tsernoglou D, Cesareni G. Genetic and structural analysis of the ColE1 Rop (Rom) protein. The EMBO Journal. 1989;8(2):621-629.
  2. ↑ Banner DW, Kokkinidis M, Tsernoglou D. Structure of the ColE1 Rop protein at 1.7 Å resolution. J Mol Biol. 1987 m.;196(3):657–75.