Difference between revisions of "Part:BBa K2259038"
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<partinfo>BBa_K2259038 short</partinfo>
 
<partinfo>BBa_K2259038 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 stabilizes 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 a full composite part of the activating RNA Trigger 1 [[part:BBa_K2259016]]. It has a constant expression by [[part:BBa_J23101]] promoter and a double terminator at the end. 
  
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This part is used together with [[part:BBa_K2259034]] to build a 3 or 4 plasmid SynORI selection gene circuit.
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Trigger 1 part activates the translation of Toehold 1 [[part:BBa_K2259014]] locked gene.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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=Introduction=
 
=Introduction=
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==Biology==
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==The overview of 4 plasmid system==
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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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[[Image:4plasmid.png|center|500px|thumb|<b>Figure 1. </b> The principle scheme of SynORI 4 plasmid selection system]]
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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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==The overview of 5 plasmid system==
  
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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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[[Image:5plasmid.png|center|500px|thumb|<b>Figure 1. </b> The principle scheme of SynORI 4 plasmid selection system]]
  
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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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== Results ==
  
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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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[[Image:5plasmidresult.jpg|center|500px|thumb|<b>Figure 1. </b> The results of 5 plasmid co-transformation using SynORI 5 plasmid selection system]]
  
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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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=About SynORI=
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==Usage with SynORI (Framework for multi-plasmid systems)==
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===About SynORI===
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[[Image:Aboutsynoritry1.png|600px|center|]]
 
[[Image:Aboutsynoritry1.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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==Toehold riboregulators in SynORI==
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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 the framework to run.  
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Toehold switches together with their corresponding RNA triggers and split antibiotic genes completes the dynamic SynORI selection system. The switches lock the translation of downstream split antibiotic genes and form an AND type gate genetic circuit which functions to stably maintain multiple plasmids in the SynORI collection.
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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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SynORI selection gene circuits for multi-plasmid systems:
  
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===Specific RNA II versions in multi-plasmid systems===
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•  2 plasmids
  
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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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Consisting of: : Two split antibiotic genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]])
  
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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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•  3 plasmids
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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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Consisting of:
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One Toehold ([[part:BBa_K2259014]] or [[part:BBa_K2259015]]),
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one Trigger RNA ([[part:BBa_K2259016]] or [[part:BBa_K2259017]]) and
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split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
  
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===Origin of RNA II biobrick===
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•  4 plasmids
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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 the 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 the SynORI framework, the wildtype ColE1 ORI is split into two different parts - <b> RNR I and RNA II </b>.
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Consisting of: Two Toeholds ([[part:BBa_K2259014]] and [[part:BBa_K2259015]]), two Trigger RNAs ([[part:BBa_K2259016]] and [[part:BBa_K2259017]]) and split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
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•  5 plasmids
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Consisting of: Modified phage control system [[part:BBa_K2259044]], two Toeholds ([[part:BBa_K2259014]] and [[part:BBa_K2259015]]), two repressed Trigger RNAs ([[part:BBa_K2259042]] and [[part:BBa_K2259043]]) and split neomycin antibiotic resistance genes ([[part:BBa_K2259018]] and [[part:BBa_K2259019]]).
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===Two groups of Toeholds===
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SynORI collection introduces two Toehold sequences termed Toehold 1 and Toehold 2 which only interact with its corresponding Trigger RNA, termed Trigger 1 and Trigger 2 and display no cross interaction.
  
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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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==Constitutive Rop protein effect on plasmid copy number==
 
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To be updated!
 
  
 
==References==
 
==References==
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<references />
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Toehold Switches: De-Novo-Designed Regulators of Gene Expression
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Green, Alexander A. et al.
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Cell, Volume 159, Issue 4, 925 - 939

Revision as of 23:07, 31 October 2017


Trigger 1 for toehold 1 activation (SynORI framework)


This is a full composite part of the activating RNA Trigger 1 part:BBa_K2259016. It has a constant expression by part:BBa_J23101 promoter and a double terminator at the end.

This part is used together with part:BBa_K2259034 to build a 3 or 4 plasmid SynORI selection gene circuit.

Trigger 1 part activates the translation of Toehold 1 part:BBa_K2259014 locked gene.

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

The overview of 4 plasmid system

Figure 1. The principle scheme of SynORI 4 plasmid selection system

The overview of 5 plasmid system

Figure 1. The principle scheme of SynORI 4 plasmid selection system

Results

Figure 1. The results of 5 plasmid co-transformation using SynORI 5 plasmid selection system

About SynORI

Aboutsynoritry1.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]!

Toehold riboregulators in SynORI

Toehold switches together with their corresponding RNA triggers and split antibiotic genes completes the dynamic SynORI selection system. The switches lock the translation of downstream split antibiotic genes and form an AND type gate genetic circuit which functions to stably maintain multiple plasmids in the SynORI collection.

SynORI selection gene circuits for multi-plasmid systems:

• 2 plasmids

Consisting of: : Two split antibiotic genes (part:BBa_K2259018 and part:BBa_K2259019)

• 3 plasmids

Consisting of: One Toehold (part:BBa_K2259014 or part:BBa_K2259015), one Trigger RNA (part:BBa_K2259016 or part:BBa_K2259017) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).

• 4 plasmids

Consisting of: Two Toeholds (part:BBa_K2259014 and part:BBa_K2259015), two Trigger RNAs (part:BBa_K2259016 and part:BBa_K2259017) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).

• 5 plasmids

Consisting of: Modified phage control system part:BBa_K2259044, two Toeholds (part:BBa_K2259014 and part:BBa_K2259015), two repressed Trigger RNAs (part:BBa_K2259042 and part:BBa_K2259043) and split neomycin antibiotic resistance genes (part:BBa_K2259018 and part:BBa_K2259019).

Two groups of Toeholds

SynORI collection introduces two Toehold sequences termed Toehold 1 and Toehold 2 which only interact with its corresponding Trigger RNA, termed Trigger 1 and Trigger 2 and display no cross interaction.



References

Toehold Switches: De-Novo-Designed Regulators of Gene Expression

Green, Alexander A. et al. Cell, Volume 159, Issue 4, 925 - 939