Difference between revisions of "Part:BBa K2918061"
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===Usage and Biology===
 
===Usage and Biology===
  
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The Φ29 replication mechanism is a unique protein-primed based replication of a linear plasmid. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, greatly simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2),terminal protein <html><a href="https://parts.igem.org/Part:BBa_K2918001"> (TP/p3)</a></html>, single stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918002"> (SSB/p5)</a></html> and double stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918003"> (DSB/p6)</a></html>.
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===Usage and Biology===
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The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (OriR and OriL), which flank the protein-primed linear plasmid <html><a href="#Nies2018">(Nies et al, 2018)</a></html>. The double stranded DNA binding proteins <html><a href="https://parts.igem.org/Part:BBa_K2918003 (DSB/p6)</a></html> aid in the process of replication and bind more intensely at the origins of replication (OriR and OriL), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases <html><a href="#Nies2018">(Nies et al, 2018)</a></html>.
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The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, do not depend on specific sequences of DNA/RNA and simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2),terminal protein <html><a href="https://parts.igem.org/Part:BBa_K2918001"> (TP/p3)</a></html>, single stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918002"> (SSB/p5)</a></html> and double stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918003"> (DSB/p6)</a></html>.
 +
The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (OriR and OriL), which flank the protein-primed linear plasmid <html><a href="#Nies2018">(Nies et al, 2018)</a></html>. The double stranded DNA binding proteins <html><a href="https://parts.igem.org/Part:BBa_K2918003"> (DSB/p6)</a></html> aid in the process of replication and bind more intensely at the origins of replication (OriR and OriL), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases <html><a href="#Nies2018">(Nies et al, 2018)</a></html>. The replication mechanism is depicted in the Figure 1.
  
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Insert Φ29 replication animation/image!
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<div><ul>
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<center>
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  <li style="display: inline-block;"> [[File:T--TUDelft--replicationpartstest.jpg|thumb|none|550px|<b>Figure 1:</b> Overview of phi 29 replication mechanism]] </li>
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</center>
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    </ul></div>
  
 
The Φ29 replication system is promising in many ways:
 
The Φ29 replication system is promising in many ways:
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<ul>
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-The Φ29 DNA Polymerase has the highest processivity of all known single subunit DNA polymerases <html><a href="#Blanco1988">(Blanco et al, 1988)</a></html>, and can be used for whole genome amplification. <br>
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<li>The Φ29 DNA Polymerase has the highest processivity of all known single subunit DNA polymerases <html><a href="#Blanco1988">(Blanco et al, 1988)</a></html>, and can be used for whole genome amplification. </li>
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-The Φ29 machinery along with cell free expression systems can be used to establish the three dogmas of biology in-vitro. Setting the basis for artificial cell development. <br>
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<li>The Φ29 machinery along with cell free expression systems can be used to establish the three dogmas of biology in-vitro. Setting the basis for artificial cell development. </li>
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-The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide  
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<li>The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide  
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sequences. <br>
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sequences. </li>
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-We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many  
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<li>We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many  
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new engineered protein-DNA complexes.<br>
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new engineered protein-DNA complexes.</li>
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-Orthogonal replication not only enables replication independent from the host, but the ability to engineer the orthogonal  
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<li>Orthogonal replication not only enables replication independent from the host, but the ability to engineer the orthogonal  
 
DNA polymerase’s fidelity without introducing mutations in the cell’s genome makes in vivo directed evolution a  
 
DNA polymerase’s fidelity without introducing mutations in the cell’s genome makes in vivo directed evolution a  
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possibility. <br>
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possibility. </li></ul>
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===Strain Construction===
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The DNA sequence of the part was synthesized by IDT and cloned by BsaI enzyme golden gate assembly in <html><a href="http://www.addgene.org/47998/pICH41308"> pICH47732 </a></html> and the sequence was confirmed by sequencing.
  
 
===Characterization===
 
===Characterization===
 
to be edited
 
to be edited
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===Strain Construction===
 
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The DNA sequence of the part was synthesized by IDT and cloned by restriction-ligation in <html><a href="http://www.addgene.org/47998/pICH41308"> pICH47732 </a></html> and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
 

Revision as of 21:18, 18 October 2019

Φ29 Right origin of replication (OriR)

Φ29 bacteriophage origin of replication (OriR)

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]

The part has been confirmed by sequencing and there are no mutations.

Usage and Biology

Usage and Biology

The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, do not depend on specific sequences of DNA/RNA and simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2),terminal protein (TP/p3), single stranded binding protein (SSB/p5) and double stranded binding protein (DSB/p6). The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (OriR and OriL), which flank the protein-primed linear plasmid (Nies et al, 2018). The double stranded DNA binding proteins (DSB/p6) aid in the process of replication and bind more intensely at the origins of replication (OriR and OriL), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases (Nies et al, 2018). The replication mechanism is depicted in the Figure 1.

  • Figure 1: Overview of phi 29 replication mechanism

The Φ29 replication system is promising in many ways:

  • The Φ29 DNA Polymerase has the highest processivity of all known single subunit DNA polymerases (Blanco et al, 1988), and can be used for whole genome amplification.
  • The Φ29 machinery along with cell free expression systems can be used to establish the three dogmas of biology in-vitro. Setting the basis for artificial cell development.
  • The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences.
  • We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many new engineered protein-DNA complexes.
  • Orthogonal replication not only enables replication independent from the host, but the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome makes in vivo directed evolution a possibility.

Strain Construction

The DNA sequence of the part was synthesized by IDT and cloned by BsaI enzyme golden gate assembly in pICH47732 and the sequence was confirmed by sequencing.

Characterization

to be edited