Difference between revisions of "Part:BBa K2259023"

 
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<partinfo>BBa_K2259023 short</partinfo>
 
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This construct defines a constitutive copy number of plasmid by using an anderson promoter to transcribe RNA I at a specific rate. RNA I controls the copy number by binding to RNA II (plasmid replication iniator) and inhibiting its ability to form primer for polymerase.
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This construct is an intermediate to 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 ([[part:BBa_K2259068]]) this device sets a defined copy number for a plasmid.  
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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_K2259023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2259023 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2259023 parameters</partinfo>
 
<partinfo>BBa_K2259023 parameters</partinfo>
 
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__TOC__
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=Introduction=
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==Biology==
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===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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<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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The interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
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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)==
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===About SynORI===
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[[Image:groupspec.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]!
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=Characterization=
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In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259068]].
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==References==
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<references />

Latest revision as of 12:52, 1 November 2017


SynORI constitutive plasmid copy number device intermediate (0.36 Anderson)

This construct is an intermediate to 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 (part:BBa_K2259068) this device sets a defined copy number for a plasmid.



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

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.

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

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_K2259068.

References