Difference between revisions of "Part:BBa K2259027"
 
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<partinfo>BBa_K2259027 short</partinfo>
 
<partinfo>BBa_K2259027 short</partinfo>
  
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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.
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This is an intermediate construct to the full SynORI constitutive copy number device. It consists of an Anderson promoter and ColE1 RNA I gene (without promoter). It can be combined with [[part:BBa_K2259000]] replication initiation part.
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When combined with RNA II this device sets a defined copy number for a plasmid (see:[[part:BBa_K2259069]]).  
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===ColE1 plasmid replication overview===
 
===ColE1 plasmid replication overview===
  
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[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. (Citation needed)]]
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[[Image:Cole1 horizontal cropped.png|300px| center|]]
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<b>ColE1-type plasmid replication begins with 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).
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<b><center>Figure 1. Main principles of ColE1 plasmid family replication</center>
  
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<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 formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).
 
  
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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.
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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>
  
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Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
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<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>
  
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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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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>
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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)==
 
==Usage with SynORI (Framework for multi-plasmid systems)==
  
 
===About SynORI===
 
===About SynORI===
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[[Image:Aboutsynoritry1.png|600px|center|]]
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[[Image:Groupspec.png|600px|center|]]
 
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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=Characterization=
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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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The two main functions of RNA II is as follows:
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# Initiating plasmid replication
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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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===Specific RNA II versions in multi-plasmid systems===
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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.
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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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===Origin of RNA II biobrick===
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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 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 SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.
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<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
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=Characterization of RNA II (Vilnius-Lithuania 2017)=
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In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259069]].
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==Constitutive Rop protein effect on plasmid copy number==
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To be updated!
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==References==
 
==References==
 
<references />
 
<references />

Latest revision as of 17:05, 1 November 2017


SynORI constitutive plasmid copy number device intermediate (0.86 Anderson)

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

When combined with RNA II this device sets a defined copy number for a plasmid (see:part:BBa_K2259069).



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

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]!

Characterization

In order to characterize this construct, it must be cloned next to RNA II gene. Please see part:BBa_K2259069.

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.