Difference between revisions of "Part:BBa K2259088"

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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|center|frame|300px|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication]]
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<b>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).
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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).
 
  
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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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<b>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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<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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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>
  
 
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]].
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This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.
 
This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.
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=Characterization of RNA II (Vilnius-Lithuania 2017)=
 
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==RNA I inactivation in wild type replicon==
 
  
 
==References==
 
==References==
 
<references />
 
<references />

Revision as of 20:16, 1 November 2017


ColE1 replicon (Deactivated RNA I, placed next to RNA II)

This is a ColE1 replicon with knocked-out RNA I promoter. A copy of RNA I is then placed next to RNA II.

This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.

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]



Introduction

Biology

ColE1 plasmid replication overview

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]!

Purpose of this construct in SynORI framework development

This construct acts as a RNA I promoter knock-out control. When RNA I promoter is disabled, plasmid copy number increases dramatically (1000-2000 copies per cell). It is difficult to tell if RNA I promoter is disabled completely because the measurements at run-away replication are not precise enough. Yet, if RNA I gene copy is placed next to knocked-out origin of replication and the plasmid copy number goes down to wildtype amounts, we can say with certainty RNA I promoter is completely disabled.

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.