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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SynORI framework gives the opportunity to have low copy plasmid groups, yet in order for them not to be lost during cell division, there must be a mechanism that actively keeps plasmids in the cell.
  
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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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[[Image:Pargif.gif|frame|400px|center| <b>Figure 1.</b> Segregation system in action]]
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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 a 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 the 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)==
  
 
===About SynORI===
 
===About SynORI===
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[[Image:Cole1 horizontal cropped.png|center|500px|thumb|<b>Figure 1. </b> Guide to SynORI - framework for multiplasmid systems. CLICK HERE TO SEE THE WHOLE COLLECTION (link needed) (Citation needed)]]
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[[Image:par.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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===Active segregation system in SynORI===
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SynORI framework gives the opportunity to have low copy plasmids, but in order for them to be stable (to not lose plasmids from the cell during cell division) there must be a mechanism that actively keeps plasmids in the cell.
 +
 +
This is where active segregation system comes in. When building a specific synthetic origin of replication, this DNA sequence can be added to stabilize the plasmid group.
  
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===Regulative RNA II molecule in SynORI===
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=Characterization of plasmid partitioning system (Vilnius-Lithuania 2017)=
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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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==Measurement of Par<sup>+</sup> and Par<sup> -</sup> pSB4A5 stability==
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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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Since we are cloning the partitioning system from pSB4A5, we must first make sure that our target sequence is ensuring the stability of pSB4A5 plasmids.
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We have deleted the partitioning sequence by using two blunt end restriction enzymes, ligating the vector and cutting the correct size vector (without the partitioning sequence) from gel after agarose electrophoresis.
  
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===Specific RNA II versions in multi-plasmid systems===
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We have then performed a stability experiment by using two plasmids - the original Par+ pSB4A5 and our Par- mutant.
  
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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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[[Image:Paras.png|thumb|900px|center|<b>Figure 2.</b>pSB4A5 plasmid copy number with and without PAR]]
  
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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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<b>Figure 2</b> - results from the first experiment where plasmid loss was evaluated using low copy BioBrick standard vector pSB4A5 + mRFP. A significant decrease in number of plasmid-containing cells is visible when active partitioning system is missing and plasmid number decrease is not visible during 100 generations period.
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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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[[Image:Paras2.png|thumb|900px|center|<b>Figure 3</b>]]
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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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Figure 3. A. Plates of transformants containing vector with active partitioning system after 20 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side. B. Plates of transformants containing vector without active partitioning system after 20 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side. C. Plates of transformants containing vector with active partitioning system after 100 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side.D. Plates of transformants containing vector without active partitioning system after 100 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side.E. After 200 generations cells were spread once again to see mRFP intensity decrease*, compared to the previous generations the number of mRFP containing colonies without active partitioning system is invisible, the intensity of mRFP of the transformants with active partitioning system is significantly decreased.
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*It is possible to evaluate plasmid-containing colonies by checking if the colony contains mRFP. However, it might be tricky since the intensity can be slightly visible in colony, in that case patching bacteria on LB plates is a more trustworthy method.
  
  
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=Characterization of RNA II (Vilnius-Lithuania 2017)=
 
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==Constitutive Rop protein effect on plasmid copy number==
 
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To be updated!
 
  
 
==References==
 
==References==
 
<references />
 
<references />

Latest revision as of 04:00, 2 November 2017


Plasmid partitioning system (SynORI framework)

SynORI framework gives the opportunity to have low copy plasmid groups, yet in order for them not to be lost during cell division, there must be a mechanism that actively keeps plasmids in the cell.

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

Figure 1. Segregation system in action

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 a 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 the 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

Par.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]!

Active segregation system in SynORI

SynORI framework gives the opportunity to have low copy plasmids, but in order for them to be stable (to not lose plasmids from the cell during cell division) there must be a mechanism that actively keeps plasmids in the cell.

This is where active segregation system comes in. When building a specific synthetic origin of replication, this DNA sequence can be added to stabilize the plasmid group.

Characterization of plasmid partitioning system (Vilnius-Lithuania 2017)

Measurement of Par+ and Par - pSB4A5 stability

Since we are cloning the partitioning system from pSB4A5, we must first make sure that our target sequence is ensuring the stability of pSB4A5 plasmids. We have deleted the partitioning sequence by using two blunt end restriction enzymes, ligating the vector and cutting the correct size vector (without the partitioning sequence) from gel after agarose electrophoresis.

We have then performed a stability experiment by using two plasmids - the original Par+ pSB4A5 and our Par- mutant.

Figure 2.pSB4A5 plasmid copy number with and without PAR

Figure 2 - results from the first experiment where plasmid loss was evaluated using low copy BioBrick standard vector pSB4A5 + mRFP. A significant decrease in number of plasmid-containing cells is visible when active partitioning system is missing and plasmid number decrease is not visible during 100 generations period.


Figure 3

Figure 3. A. Plates of transformants containing vector with active partitioning system after 20 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side. B. Plates of transformants containing vector without active partitioning system after 20 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side. C. Plates of transformants containing vector with active partitioning system after 100 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side.D. Plates of transformants containing vector without active partitioning system after 100 generations. LB plates without antibiotic - left side. LB plates with ampicillin - right side.E. After 200 generations cells were spread once again to see mRFP intensity decrease*, compared to the previous generations the number of mRFP containing colonies without active partitioning system is invisible, the intensity of mRFP of the transformants with active partitioning system is significantly decreased.

  • It is possible to evaluate plasmid-containing colonies by checking if the colony contains mRFP. However, it might be tricky since the intensity can be slightly visible in colony, in that case patching bacteria on LB plates is a more trustworthy method.


References