Difference between revisions of "Part:BBa K2259067"
 
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<partinfo>BBa_K2259067 short</partinfo>
 
<partinfo>BBa_K2259067 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 device is a fully functional synthetic origin of replication that sets a constitutive copy number to a plasmid. Different concentrations of RNA I gene provide a different copy number of a plasmid.  
 +
 
 +
Devices from the same series that have different Anderson promoters: [[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson), [[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson).
  
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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!
 
  
 
See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
 
See how this part fits into the whole SynORI framework [[#About SynORI|by pressing here!]]
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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)]]
 
[[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>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 formation of a secondary structure of RNA II that is necessary for hybridization of RNA II to the template DNA (Figure 1. B).
+
<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).
  
 
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.
 
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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The interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
  
 
==Usage with SynORI (Framework for multi-plasmid systems)==
 
==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:Groupspec.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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===This device 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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This is a constitutive copy number device which sets a specific copy number for a plasmid. These constitutive devices can be used with different Anderson promoters to select a different copy number.
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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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 +
Devices from the same series that have different Anderson promoters: [[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson),[[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson).
  
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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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See the [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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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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===Further details===
 +
For more background information and indepth insight on this part's design please see the individual part pages of [[part:BBa_K2259000]] and [[part:BBa_K2259005]].
  
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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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===Origin of RNA II biobrick===
 
  
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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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=Characterization of RNA II (Vilnius-Lithuania 2017)=
 +
==Interaction between RNA I and RNA II groups==
 +
===Constitutive promoter===
  
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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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Once the RNA I promoter was [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2259000#RNA_I_inactivation_in_wild_type_replicon disabled] in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit. We have discovered the sequence of wild type RNA I promoter by using PromoterHunter and removed it, thus creating a wild type RNA I gene [[part:BBa_K2259005]]. First, series of Anderson promoters were cloned next to the RNA I gene ([[part:BBa_K2259021]] (0.15 Anderson), [[part:BBa_K2259023]] (0.36 Anderson), [[part:BBa_K2259027]] (0.86 Anderson), [[part:BBa_K2259028]] (1.0 Anderson)) and then placed next to RNA II ([[part:BBa_K2259067]] (0.15 Anderson), [[part:BBa_K2259068]] (0.36 Anderson), [[part:BBa_K2259069]] (0.86 Anderson), [[part:BBa_K22590671]] (1.0 Anderson)).
  
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<Picture of how RNA I promoter mutations might destroy RNA II secondary structure.>
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[[File:RNA_I_anderson.png|thumb|centre|900px|<b>Figure 4. </b> RNA I and RNA II constructs, with RNA I constructs under different-strength Anderson promoters.]]
 +
 
 +
In theory (see “Modelling” at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]), lower-strength Anderson promoters should yield lower concentrations of RNA I, hence higher copy numbers of plasmids per cell.  Our constitutive copy number device experiment results prove it to be true in practice as well. The stronger Anderson promoter is used, the less copy number per cell we get. With the strongest Anderson we get only 21+-6.84 plasmids per cell.
 +
 
 +
Worth to mention is that the closest to wild type ColE1 replicon is the 0.86 strength Anderson promoter ([[Part:BBa_J23102]]), measured by copy number alone.
 +
 
 +
We can state with certainty that we are now able to control the plasmid copy number in a constitutive manner, and we simply call it the SynORI constitutive copy number device.
 +
 
 +
===Inducible promoter===
 +
 
 +
We wanted to move one step further and try to build an inducible copy number system. We first had to make sure that at least part of our construct is well characterized and to do so we chose the Rhamnose promoter from the biobrick registry ([[Part:BBa_K914003]])
 +
 
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For this experiment we have built a Rhamnose promoter and RNA I construct [[part:BBa_K2259065]] and then cloned this construct next to RNA II [[part:BBa_K2259091]]. We have used different concentration of Rhamnose in our media in order to see if this approach was possible and if so, to figure out the dependency between the plasmid copy number and rhamnose concentration.
 +
 
 +
[[File:Rha_rnr.png|thumb|centre|900px|<b>Figure 5. </b> RNA I and RNA II constructs, with RNA I gene being under the Rhamnose promoter, inducided by different rhamnose concentrations.]]
 +
 
 +
The first thing we noticed was that Rhamnose promoter was very strong in terms of plasmid copy number reduction. It was also considerably leaky (promoter can be enabled even without any inducer). At zero induction there were approximately only 9 plasmids per cell and at 1 percent induction the number dropped to approximately 1 plasmid per cell.
 +
 
 +
RNA I rhamnose-induced promoter seemed to be working well, with higher concentrations of inductor giving lower plasmid copy number.
 +
 
 +
We called it the SynORI copy number induction device.
 +
 
 +
So now when we can flexibly control the copy number of a plasmids, the only question is - what will come next?
  
−
=Characterization of RNA II (Vilnius-Lithuania 2017)=
 
−
==RNA I inactivation in wild type replicon==
 
  
 
==References==
 
==References==
 
<references />
 
<references />

Latest revision as of 23:42, 1 November 2017


SynORI constitutive plasmid copy number device (0.15)

This device is a fully functional synthetic origin of replication that sets a constitutive copy number to a plasmid. Different concentrations of RNA I gene provide a different copy number of a plasmid.

Devices from the same series that have different Anderson promoters: part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson), part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson).


See how this part fits into the whole SynORI framework by pressing here!


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 685
    Illegal NheI site found at 708
  • 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.

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]!

This device in SynORI

This is a constitutive copy number device which sets a specific copy number for a plasmid. These constitutive devices can be used with different Anderson promoters to select a different copy number.

Devices from the same series that have different Anderson promoters: part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson),part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson).


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

Further details

For more background information and indepth insight on this part's design please see the individual part pages of part:BBa_K2259000 and part:BBa_K2259005.


Characterization of RNA II (Vilnius-Lithuania 2017)

Interaction between RNA I and RNA II groups

Constitutive promoter

Once the RNA I promoter was disabled in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit. We have discovered the sequence of wild type RNA I promoter by using PromoterHunter and removed it, thus creating a wild type RNA I gene part:BBa_K2259005. First, series of Anderson promoters were cloned next to the RNA I gene (part:BBa_K2259021 (0.15 Anderson), part:BBa_K2259023 (0.36 Anderson), part:BBa_K2259027 (0.86 Anderson), part:BBa_K2259028 (1.0 Anderson)) and then placed next to RNA II (part:BBa_K2259067 (0.15 Anderson), part:BBa_K2259068 (0.36 Anderson), part:BBa_K2259069 (0.86 Anderson), part:BBa_K22590671 (1.0 Anderson)).

Figure 4. RNA I and RNA II constructs, with RNA I constructs under different-strength Anderson promoters.

In theory (see “Modelling” at [http://2017.igem.org/Team:Vilnius-Lithuania team Vilnius-Lithuania wiki]), lower-strength Anderson promoters should yield lower concentrations of RNA I, hence higher copy numbers of plasmids per cell. Our constitutive copy number device experiment results prove it to be true in practice as well. The stronger Anderson promoter is used, the less copy number per cell we get. With the strongest Anderson we get only 21+-6.84 plasmids per cell.

Worth to mention is that the closest to wild type ColE1 replicon is the 0.86 strength Anderson promoter (Part:BBa_J23102), measured by copy number alone.

We can state with certainty that we are now able to control the plasmid copy number in a constitutive manner, and we simply call it the SynORI constitutive copy number device.

Inducible promoter

We wanted to move one step further and try to build an inducible copy number system. We first had to make sure that at least part of our construct is well characterized and to do so we chose the Rhamnose promoter from the biobrick registry (Part:BBa_K914003)

For this experiment we have built a Rhamnose promoter and RNA I construct part:BBa_K2259065 and then cloned this construct next to RNA II part:BBa_K2259091. We have used different concentration of Rhamnose in our media in order to see if this approach was possible and if so, to figure out the dependency between the plasmid copy number and rhamnose concentration.

Figure 5. RNA I and RNA II constructs, with RNA I gene being under the Rhamnose promoter, inducided by different rhamnose concentrations.

The first thing we noticed was that Rhamnose promoter was very strong in terms of plasmid copy number reduction. It was also considerably leaky (promoter can be enabled even without any inducer). At zero induction there were approximately only 9 plasmids per cell and at 1 percent induction the number dropped to approximately 1 plasmid per cell.

RNA I rhamnose-induced promoter seemed to be working well, with higher concentrations of inductor giving lower plasmid copy number.

We called it the SynORI copy number induction device.

So now when we can flexibly control the copy number of a plasmids, the only question is - what will come next?


References