Difference between revisions of "Part:BBa K2259011"

(→‎Active segregation system in SynORI)
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=Introduction=
 
=Introduction=
 
==Biology==
 
==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.
+
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 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).  
+
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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===Active segregation system in SynORI===
 
===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 cell during cell division) there must be a mechanism that actively keeps plasmids in the cell.
+
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 stabilise the plasmid group.
+
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)=
 
=Characterization of plasmid partitioning system (Vilnius-Lithuania 2017)=
 
==Measurement of Par<sup>+</sup> and Par<sup> -</sup> pSB4A5 stability==
 
==Measurement of Par<sup>+</sup> and Par<sup> -</sup> 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.  
 
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 cuting the correct size vector (without the partitioning sequence) from gel after agarose electrophoresis.  
+
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.
 
We have then performed a stability experiment by using two plasmids - the original Par+ pSB4A5 and our Par- mutant.

Revision as of 16:28, 31 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 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

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]!

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.

<IMAGE of plates>, protein expression differences

References