Difference between revisions of "Part:BBa K2259011"
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<partinfo>BBa_K2259011 short</partinfo>
 
<partinfo>BBa_K2259011 short</partinfo>
  
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This DNA sequence acts a regulatory site for plasmid stabilization proteins to bind. Stabilization proteins are coded by E. Coli thus are not needed as separate parts in plasmid.
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This particular biobrick encodes low-copy plasmid partitioning system, designed specifically to stabilize SynORI (multi-plasmid system, read more here <link>) low copy plasmid groups, but can also be used to stabilize any type of plasmid.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2259011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2259011 SequenceAndFeatures</partinfo>
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=Introduction=
 
=Introduction=
 
==Biology==
 
==Biology==
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===ColE1 plasmid replication overview===
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Par region derived from pSC101 is a lot different from its counterparts, for example F and P1 plasmids (Meacock and Cohen 1980). It does not seem to encode any protein but contains binding site for DNA gyrase (Wahle and Kornberg 1988). In contrast, both F and P1 systems encode partitioning proteins in plasmids making Par regions as big as 2kb.
  
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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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Plasmids with partial deletions of par in pSC101 have decreased negative supercoiling and are extremely unstable (lost from cells in a short amount of time). This has led to proposal that gyrase-generated negative supercoiling establishes a DNA conformation which enables plasmids to interact with E. Coli structures which distribute them to daughter cells at division (Miller et al., 1990).  
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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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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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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)==
 
==Usage with SynORI (Framework for multi-plasmid systems)==
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===Regulative RNA II molecule in SynORI===
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===Active segregation system 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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SynORI system can be used with low plasmid systems, but in order to efficiently run that kind of system, it must have a mechanism to keep low copy plasmids in the cell.
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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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===Specific RNA II versions in multi-plasmid systems===
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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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<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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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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===Origin of RNA II biobrick===
 
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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 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 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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=Characterization of Active segregation system (Vilnius-Lithuania 2017)=
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==Constitutive Rop protein effect on plasmid copy number==
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To be updated!
 
To be updated!
  
 
==References==
 
==References==
 
<references />
 
<references />

Revision as of 21:47, 24 October 2017


Plasmid partitioning system (SynORI framework)

This particular biobrick encodes low-copy plasmid partitioning system, designed specifically to stabilize SynORI (multi-plasmid system, read more here <link>) low copy plasmid groups, but can also be used to stabilize any type of plasmid.


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

Biology

Par region derived from pSC101 is a lot different from its counterparts, for example F and P1 plasmids (Meacock and Cohen 1980). It does not seem to encode any protein but contains binding site for DNA gyrase (Wahle and Kornberg 1988). In contrast, both F and P1 systems encode partitioning proteins in plasmids making Par regions as big as 2kb.

Plasmids with partial deletions of par in pSC101 have decreased negative supercoiling and are extremely unstable (lost from cells in a short amount of time). This has led to proposal that gyrase-generated negative supercoiling establishes a DNA conformation which enables plasmids to interact with E. Coli structures which distribute them to daughter cells at division (Miller et al., 1990).


Usage with SynORI (Framework for multi-plasmid systems)

About SynORI

Figure 1. Guide to SynORI - framework for multiplasmid systems. CLICK HERE TO SEE THE WHOLE COLLECTION (link needed) (Citation needed)

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]!


Active segregation system in SynORI

SynORI system can be used with low plasmid systems, but in order to efficiently run that kind of system, it must have a mechanism to keep low copy plasmids in the cell.




Characterization of Active segregation system (Vilnius-Lithuania 2017)

To be updated!

References