Difference between revisions of "Part:BBa K2259081"
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<partinfo>BBa_K2259081 short</partinfo>
 
<partinfo>BBa_K2259081 short</partinfo>
  
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RNA II acts as a plasmid replication initiatior. The transcript folds into a secondary structure which stabilises the interaction between the nascent RNA and the plasmids DNA. This RNA-DNA 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 and consequently, the start of plasmid replication.
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This is the updated version of iGEM base vector [[Part:BBa_I51020]] that has been withdrawn from the database due to encoding a ccdB toxin. Compared to BBa_I51020 the ccdB toxin was switched out to blue and white selection for picking colonies with the replaced origin of replication. [[Part:BBa_I51020]] page can still be used as a reliable source for this part's design and function.
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*Caution! <B>RNA II (Group A)</b> indicates that this plasmid only interacts with regulatory <B>RNA I (Group A)</b> <LINK TO RNA I A> from SynORI (framework for multiplasmid systems) collection and is stable when placed with other SynORI plasmid groups. RNA II A will not be regulated with RNA I from another group!
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See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
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<partinfo>BBa_K2259081 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2259081 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
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<partinfo>BBa_K2259081 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 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 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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Interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
 
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==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:synori.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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==Base vector 2.0 in SynORI framework==
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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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[[File:collect.png|950px]]
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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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=== 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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Engineering an improved, functional base vector 2.0 is crucial for the SynORI framework.  
  
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===Origin of RNA II biobrick===
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Building a modular a synthetic origin of replication is easiest when working in biobrick region. But for synthetic ORI this is only possible if there is no other origin of replication in the rest of the vector. Base vector 2.0 provides this, as it has its pUC replicon in biobrick site.
  
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If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?
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One can then replace the pUC origin of replication to SynORI system parts. Once the modular SynORI system is built, it can be transferred to another plasmid location and biobricks are then free to use for any task.
  
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In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in ColE1 origin of replication. That, however, was not a trivial task, because by changing RNA I promoter sequence, one also changes the RNA II secondary structure, which is crucial for plasmid replication initiation. 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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See [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).
  
 
=Characterization of RNA II (Vilnius-Lithuania 2017)=
 
=Characterization of RNA II (Vilnius-Lithuania 2017)=

Latest revision as of 12:44, 1 November 2017


Base vector 2.0 - a backbone for SynORI multi-plasmid framework

This is the updated version of iGEM base vector Part:BBa_I51020 that has been withdrawn from the database due to encoding a ccdB toxin. Compared to BBa_I51020 the ccdB toxin was switched out to blue and white selection for picking colonies with the replaced origin of replication. Part:BBa_I51020 page can still be used as a reliable source for this part's design and function.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 968
    Illegal suffix found at 2107
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 968
    Illegal NheI site found at 745
    Illegal NheI site found at 2283
    Illegal SpeI site found at 2108
    Illegal PstI site found at 2122
    Illegal NotI site found at 974
    Illegal NotI site found at 2115
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 968
    Illegal BglII site found at 1451
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 968
    Illegal suffix found at 2108
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 968
    Plasmid lacks a suffix.
    Illegal XbaI site found at 983
    Illegal SpeI site found at 2108
    Illegal PstI site found at 2122
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


About SynORI

Synori.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]!

Base vector 2.0 in SynORI framework

Collect.png


Engineering an improved, functional base vector 2.0 is crucial for the SynORI framework.

Building a modular a synthetic origin of replication is easiest when working in biobrick region. But for synthetic ORI this is only possible if there is no other origin of replication in the rest of the vector. Base vector 2.0 provides this, as it has its pUC replicon in biobrick site.

One can then replace the pUC origin of replication to SynORI system parts. Once the modular SynORI system is built, it can be transferred to another plasmid location and biobricks are then free to use for any task.


See [http://2017.igem.org/Team:Vilnius-Lithuania Vilnius-Lithuania 2017 team wiki] for more insight about our synthetic origin of replication (SynORI).

Characterization of RNA II (Vilnius-Lithuania 2017)

RNA I inactivation in wild type replicon

References