Difference between revisions of "Part:BBa K2259000"

 
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RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilises 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.
 
RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilises 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.
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2259000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2259000 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2259000 parameters</partinfo>
 
<partinfo>BBa_K2259000 parameters</partinfo>
 
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__TOC__
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[[Image:Scheme of rop.jpeg|right|225px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)]]
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=Introduction=
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==Biology==
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===ColE1 replication===
 +
TestTest
 +
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===Test test===
 +
testest
 +
 +
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[[Image:Rop protein 3d small.gif|right|500px|frame|<b>Figure 2. </b>Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.<ref>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.</ref>]]
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Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).
 +
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==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]!
 +
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===Regulative RNA II molecule in SynORI===
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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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<b>For example:</b> 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
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protein can be used to <b>globally lower the copy number of each group </b> - 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)=
 +
==Constitutive Rop protein effect on plasmid copy number==
 +
To be updated!
 +
 +
==References==
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<references />

Revision as of 13:07, 20 October 2017


SynORI framework RNA II - Replication Initiator (Group A)

RNAII acts as a pre-primer and begins the synthesis of plasmid DNA leader strand. The transcript folds into a secondary structure which stabilises 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.


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

ColE1 replication

TestTest

Test test

testest


Figure 2. Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.[1]

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

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

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)

Constitutive Rop protein effect on plasmid copy number

To be updated!

References

  1. ↑ 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.