Difference between revisions of "Part:BBa K2259084"
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<partinfo>BBa_K2259084 short</partinfo>
 
<partinfo>BBa_K2259084 short</partinfo>
  
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RNA II acts as a plasmid replication initiator. The transcript folds into a secondary structure which stabilizes 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 the same part as [[part:BBa_K2259038]] but it has no upstream promoter
  
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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 multi-plasmid 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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This is a composite part (without promoter) of the activating RNA Trigger 1 [[part:BBa_K2259016]]. It has a double terminator at the end. 
  
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See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
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This part (when coupled with a promoter) 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 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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==Usage with SynORI (Framework for multi-plasmid systems)==
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=About SynORI=
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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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=== RNA II and RNA I in the engineering of unique plasmid  groups for multi-plasmid system===
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•  2 plasmids
  
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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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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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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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•  4 plasmids
  
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===Origin of RNA II biobrick===
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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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If RNA II and RNA I are naturally an antisense system, why are there two separate constructs in SynORI system?
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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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In order to flexibly control the synthesis of RNA I, the RNA I gene first needed to be inactivated in the 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 the 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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==RNA I inactivation in wild type replicon==
 
  
 
==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 23:13, 31 October 2017


Intermediate of Trigger 1 for toehold 1 activation (SynORI framework)

This the same part as part:BBa_K2259038 but it has no upstream promoter

This is a composite part (without promoter) of the activating RNA Trigger 1 part:BBa_K2259016. It has a double terminator at the end.

This part (when coupled with a promoter) 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
    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

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