Difference between revisions of "Part:BBa K2259073"
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This construct is an intermediate to full SynORI global copy number control device. It consists of an Anderson promoter, Anderson RBS, ROP protein and a double terminator.
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This construct is an intermediate to full SynORI global copy number control device. It consists of a weak Anderson promoter, weak Anderson RBS, ROP protein and a double terminator.
 +
 
 +
When placed in a SynORI system this device lowers plasmid copy number of each group as it can bypass the selective control.
 +
 
 +
Device also works with other plasmids consisting of ColE1 origin of replication
 +
 
 +
Devices from the same series that have different Anderson promoters:  [[part:BBa_K2259072]] (0 Anderson), [[part:BBa_K2259073]] (0.15 Anderson), [[part:K2259074]] (0.24 Anderson).
  
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When combined with RNA II ([[part:BBa_K2259068]]) this device lowers plasmid copy number of each plasmid group in the system.
 
  
  
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[[Image:Scheme of rop.jpeg|right|225px|thumb|<b>Figure 1. </b> Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)]]
  
 
=Introduction=
 
=Introduction=
 
==Biology==
 
==Biology==
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===ColE1 plasmid replication overview===
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<b>Repressor of primer (ROP)</b> is a small dimeric protein that participates in ColE1 plasmid family copy number control, by increasing affinity between two complementary RNAs - RNA I (Replication inhibitor) and RNA II (Replication activator) (Fig. 1). <ref>Castagnoli L, Scarpa M, Kokkinidis M, Banner DW, Tsernoglou D, Cesareni G. Genetic and structural analysis of the ColE1 Rop (Rom) protein. The EMBO Journal. 1989;8(2):621-629.</ref> By increasing affinity of the two RNA molecules, Rop decreases the rate of plasmid replication initiation events.
  
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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:Rop protein 3d small.gif|right|500px|frame|<b>Figure 2. </b>Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.<ref>Banner DW, Kokkinidis M, Tsernoglou D. Structure of the ColE1 Rop protein at 1.7 Å resolution. J Mol Biol. 1987 m.;196(3):657–75.</ref>]]
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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 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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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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The interaction between RNA I and RNA II can be amplified by Rop protein, see [[part:BBa_K2259010]].
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Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).
 
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===
 
===About SynORI===
 
[[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]!
  
 +
===This device in SynORI===
 +
This is a constitutive global copy number modulator device which lowers plasmid copy number of every group in the system bypassing the selective control of different groups. 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_K2259072]] (0 Anderson), [[part:BBa_K2259073]] (0.15 Anderson), [[part:K2259074]] (0.24 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===
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For more background information and indepth insight on this part's design please see the individual part page of ROP protein[[part:BBa_K2259010]].
  
 
=Characterization=
 
=Characterization=
  
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In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259053]].
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In order to characterize this construct, it must be cloned next to RNA II gene. Please see [[part:BBa_K2259052]].
  
 
==References==
 
==References==
 
<references />
 
<references />

Revision as of 12:40, 1 November 2017


Rop protein with anderson (0.15) promoter for global plasmid copy number control


This construct is an intermediate to full SynORI global copy number control device. It consists of a weak Anderson promoter, weak Anderson RBS, ROP protein and a double terminator.

When placed in a SynORI system this device lowers plasmid copy number of each group as it can bypass the selective control.

Device also works with other plasmids consisting of ColE1 origin of replication

Devices from the same series that have different Anderson promoters: part:BBa_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 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 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]



Figure 1. Main principles of ColE1 plasmid family replication. Rop protein interaction region marked in red square. (Citation needed)

Introduction

Biology

Repressor of primer (ROP) is a small dimeric protein that participates in ColE1 plasmid family copy number control, by increasing affinity between two complementary RNAs - RNA I (Replication inhibitor) and RNA II (Replication activator) (Fig. 1). [1] By increasing affinity of the two RNA molecules, Rop decreases the rate of plasmid replication initiation events.

Figure 2. Structure of the ColE1 Rop protein, at 1.7 angstroms resolution.[2]

Rop dimer is a bundle of four tightly packed alpha helices that are held by hydrophobic interactions (Fig. 2).

Usage with SynORI (Framework for multi-plasmid systems)

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]!

This device in SynORI

This is a constitutive global copy number modulator device which lowers plasmid copy number of every group in the system bypassing the selective control of different groups. 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_K2259072 (0 Anderson), part:BBa_K2259073 (0.15 Anderson), part:K2259074 (0.24 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 page of ROP proteinpart:BBa_K2259010.

Characterization

In order to characterize this construct, it must be cloned next to RNA II gene. Please see part:BBa_K2259052.

References

  1. ↑ Castagnoli L, Scarpa M, Kokkinidis M, Banner DW, Tsernoglou D, Cesareni G. Genetic and structural analysis of the ColE1 Rop (Rom) protein. The EMBO Journal. 1989;8(2):621-629.
  2. ↑ Banner DW, Kokkinidis M, Tsernoglou D. Structure of the ColE1 Rop protein at 1.7 Å resolution. J Mol Biol. 1987 m.;196(3):657–75.