Difference between revisions of "Part:BBa K2259083"
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<partinfo>BBa_K2259083 short</partinfo>
 
<partinfo>BBa_K2259083 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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intermediate of the full composite part [[part:BBa_K2259034]] - 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 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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A composite part of the riboregulatory sequence Toehold 2 [[part:BBa_K2259015]] coupled with the beta subunit of the split antibiotic resistance gene [[Part:BBa_K2259019]]. 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 (with upstream promoter) is used together with [[part:BBa_K2259040]] to build a 3 or 4 plasmid SynORI selection gene circuit. Additionally, it can be used with signal activated trigger [[part:BBa_K2259043]] to build a 5 plasmid SynORI selection system
  
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Toehold 2 part is activated by the Trigger 2 [[part:BBa_K2259017]]
  
 
<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:4plasmid.png|center|500px|thumb|<b>Figure 1. </b> The schematic representation of 4 plasmid SynORI selection system. First 2 plasmids constantly express Toehold 1 and 2 which are coupled with the split antibiotic alpha and beta subunits but has its translation locked. Trigger 1 and 2 are constantly expressed by other 2 to plasmids to unlock the resistance gene translation. If any of the plasmid is lost, the cell dies.]]
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==The overview of 5 plasmid system==
  
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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:5plasmid.png|center|500px|thumb|<b>Figure 1. </b> The schematic representation of 5 plasmid SynORI selection system. First plasmid constantly expresses lambda, the activator of the modified phage promoter, which controls the expression of the Trigger 1 and 2. The Triggers unlock the translation of split resistance gene controlled by Toehold 1 and 2. Additionally, 434 repressor is constantly expressed to regulate the modified phage promoter. If any of the plasmid is lost, the cell dies.]]
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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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== Results ==
  
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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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[[Image:5plasmidresult.jpg|center|500px|thumb|<b>Figure 1. </b> SynORI 5 plasmid co-transformation results 1 - No trigger 1 (control). 2 - No trigger 2 (control). 3 - No lambda activator plasmid (control). 4 - Full System: lambda activator plasmid; toehold 1 alpha-neo; toehold 2 beta-neo; trigger 1; trigger 2]]
  
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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)==
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=About SynORI=
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[[Image:Sel.png|600px|center|]]
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===About SynORI===
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[[Image:Aboutsynoritry1.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]!
 
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 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 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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=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

Latest revision as of 13:30, 1 November 2017


Intermediate of Toehold activated beta-neomycin (SynORI framework)

intermediate of the full composite part part:BBa_K2259034 - it has no upstream promoter

A composite part of the riboregulatory sequence Toehold 2 part:BBa_K2259015 coupled with the beta subunit of the split antibiotic resistance gene Part:BBa_K2259019. It has a double terminator at the end.

This part (with upstream promoter) is used together with part:BBa_K2259040 to build a 3 or 4 plasmid SynORI selection gene circuit. Additionally, it can be used with signal activated trigger part:BBa_K2259043 to build a 5 plasmid SynORI selection system

Toehold 2 part is activated by the Trigger 2 part:BBa_K2259017

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 541
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 390
    Illegal SapI.rc site found at 600



Introduction

The overview of 4 plasmid system

Figure 1. The schematic representation of 4 plasmid SynORI selection system. First 2 plasmids constantly express Toehold 1 and 2 which are coupled with the split antibiotic alpha and beta subunits but has its translation locked. Trigger 1 and 2 are constantly expressed by other 2 to plasmids to unlock the resistance gene translation. If any of the plasmid is lost, the cell dies.

The overview of 5 plasmid system

Figure 1. The schematic representation of 5 plasmid SynORI selection system. First plasmid constantly expresses lambda, the activator of the modified phage promoter, which controls the expression of the Trigger 1 and 2. The Triggers unlock the translation of split resistance gene controlled by Toehold 1 and 2. Additionally, 434 repressor is constantly expressed to regulate the modified phage promoter. If any of the plasmid is lost, the cell dies.

Results

Figure 1. SynORI 5 plasmid co-transformation results 1 - No trigger 1 (control). 2 - No trigger 2 (control). 3 - No lambda activator plasmid (control). 4 - Full System: lambda activator plasmid; toehold 1 alpha-neo; toehold 2 beta-neo; trigger 1; trigger 2


About SynORI

Sel.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