Difference between revisions of "Part:BBa K2918018"
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<partinfo>BBa_K2918018 short</partinfo>
 
<partinfo>BBa_K2918018 short</partinfo>
  
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A level 1 MoClo construct with a 0.5 T7 promoter, a universal RBS, TP and a T7 terminator
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A level 1 MoClo construct with a medium T7 promoter, a universal RBS, phi29 TP and a T7 terminator
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2918018 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2918018 SequenceAndFeatures</partinfo>
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The construct is confirmed by sequencing and there are no mutations.
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===Usage and Biology===
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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, does not depend on specific sequences of DNA/RNA which simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase <html><a href="https://parts.igem.org/Part:BBa_K2918034">(DNAP/p2)</a></html>, 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 (<html><a href="https://parts.igem.org/Part:BBa_K2918033">OriL</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918061">OriR</a></html>), which flank the protein-primed linear plasmid <html><a href="#Salas1994">(Salas et al., 1994)</a></html>. The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), 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="#Salas1994">(Salas et al., 1994)</a></html>. The replication mechanism is depicted in Figure 1.  If you want to read more about this mechanism, you can take a look at our <html><a target="_blank" href="http://2019.igem.org/Team:TUDelft/Design#orthorep">Design</a></html> page!
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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 phi29 replication mechanism]] </li>
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</center>
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    </ul></div>
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The Φ29 replication system is promising in many ways:
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<ul>
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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., 1989)</a></html>, and can be used for whole genome amplification. </li>
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<li>The Φ29 machinery along with cell-free expression systems can be used to establish the three dogmas of biology <I>in vitro</I>, setting the basis for artificial cell development. </li>
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<li>The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide
 +
sequences. </li>
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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
 +
new engineered protein-DNA complexes.</li>
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<li>Orthogonal replication enables not only replication independent from the host, but also the ability to engineer the orthogonal
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DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes <I>in vivo</I> directed evolution a
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possibility. </li></ul>
  
 
===Toxicity===
 
===Toxicity===
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<div><ul>  
 
<div><ul>  
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   <li style="display: inline-block;"> [[File:T--TUDelft--0.5tp-nomM.png|thumb|none|444px|<b>Figure 3A:</b> The growth curve of TP under a 0.5 T7 promoter in hours with 1 mM IPTG induction]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--TPnoiptg.png|thumb|none|444px|<b>Figure 2A:</b> Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--0.5tp-1mM.png|thumb|none|444px|<b>Figure 3B:</b> The growth curve of TP under a 0.5 T7 promoter in hours with 1 mM IPTG induction]] </li>
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     <li> [[File:T--TUDelft--0.5tp-10mM.png|thumb|center|444px|<b>Figure 3C:</b> The growth curve of TP under a 0.5 T7 promoter in hours with 10 mM IPTG inductionn]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--tp1iptg.png|thumb|none|444px|<b>Figure 2B:</b> Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction]] </li>
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     <li> [[File:T--TUDelft--tp10iptg.png|thumb|center|444px|<b>Figure 2C:</b> Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction]] </li>
 
     </ul></div>
 
     </ul></div>
  
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<!-- Add more about the biology of this part here
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===References===
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===Usage and Biology===
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<html>
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<ul>
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<li>
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<a id="Salas1994" href="https://www.pnas.org/content/91/25/12198" target="_blank">
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Blanco, L., Lázaro, J. M., De Vega, M., Bonnin, A., & Salas, M. (1994). Terminal protein-primed DNA amplification.<I> Proceedings of the National Academy of Sciences of the United States of America</I>.</a>
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</li>
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<li>
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<a id="Blanco1988" href="https://www.ncbi.nlm.nih.gov/pubmed/2498321" target="_blank">
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Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.</a>
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</li>
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</ul>
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</html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 06:23, 14 December 2019


Medium T7 promoter - Universal RBS - Φ29 TP - T7 terminator

A level 1 MoClo construct with a medium T7 promoter, a universal RBS, phi29 TP and a T7 terminator

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 237
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 557
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

The construct is confirmed by sequencing and there are no mutations.

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, does not depend on specific sequences of DNA/RNA which 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 (OriL and OriR), which flank the protein-primed linear plasmid (Salas et al., 1994). The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), 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 (Salas et al., 1994). The replication mechanism is depicted in Figure 1. If you want to read more about this mechanism, you can take a look at our Design page!

  • Figure 1: Overview of phi29 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., 1989), 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 enables not only replication independent from the host, but also the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes in vivo directed evolution a possibility.

Toxicity

Our Sci-Phi 29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in E. coli BL21(DE3) pLysS. The results are shown in the graphs below.

  • Figure 2A: Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction
  • Figure 2B: Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction
  • Figure 2C: Normalized maximum growth rate of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction

References