Difference between revisions of "Part:BBa K2918061"
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<partinfo>BBa_K2918061 short</partinfo>
 
<partinfo>BBa_K2918061 short</partinfo>
  
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<span class='h3bb'>Sequence and Features</span>
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Φ29 bacteriophage origin of replication (OriR)
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<partinfo>BBa_K2918061 SequenceAndFeatures</partinfo>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span><partinfo>BBa_K2918061 SequenceAndFeatures</partinfo>
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Many promoter parts contain sequences downstream of the Transcription Start Site (such as operators or assembly fusion sites) resulting in extra unintended sequences in the transcript. These additional sequences are shown to significantly affect gene expression levels, disrupting the modularity and predictability of synthetic parts <html><a href="#Lou2012">(Lou et al., 2012)</a></html>). Therefore, to insulate the translation rates of the part from the use of different promoters, ribozymes can be used for their self cleavage properties and remove these sequences upstream of mRNA. By the inclusion of ribozymes in 5’ UTR parts, outputs of genetic circuits will be insulated from genetic context, but the presence of multiple copies of the same ribozyme in different genes may result in homologous recombination <html><a href="#Lou2012">(Lou et al., 2012)</a></html>). With that in mind, we, from TU Delft 2019, have designed Type IIS parts of both ribozymes and RBS for modular assembly in any combination desired.
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Unfortunately, junction sequences such as Type IIS overhangs between ribozyme and RBS can also influence translation rates. To achieve scarless modular cloning of ribozymes and RBS, the strategy illustrated below can be adopted.  
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The part has been confirmed by sequencing and there are no mutations.
  
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===Usage and Biology===
  
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Lou et al have screened and identified a series of ribozymes for insulating genetic circuits. All of these ribozymes contain conserved 3’ ends  (ACCTCTACAAATAATTTT<b>GTTT</b>AA) and the 4 highlight nucleotides can be used as a fusion site identified as compatible to Type IIS cloning. In the RBS sequence, AA should be added upstream in order to complement the incomplete ribozyme sequence when assembled.
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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>
  
 
===Strain Construction===
 
===Strain Construction===
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The DNA sequence of the part was synthesized by IDT with flanking BsaI sites and AATG 3' overhang.  The RBS was then cloned along with an altered ribozyme [https://parts.igem.org/Part:BBa_K2918012/ RiboJ] in a level 0 MoClo backbone [http://www.addgene.org/47992/ pICH41246] and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning section below. Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html> for the detailed protocol.  
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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 target="_blank" href="http://www.addgene.org/47998"> pICH47732 </a></html> and the sequence was confirmed by sequencing.
  
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===Characterization===
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To characterize our linear construct we wanted to demonstrate <i>in vitro</i> replication of our linear construct <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2918029">(OriL-GFP-Kan-OriR)</a></html> and show that our construct can be propagated by the phi29 replication machinery.
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We used an <I>in vitro</I> transcription-translation system (PUREfrex®2.0) supplemented with dNTPs and purified auxiliary proteins: SSB and DSB, needed for an efficient DNA replication. We used 1nM as the starting DNA concentration of our OriL-GFP-Kan-OriR with a twofold excess of plasmid DNA encoding for both DNAP and TP. After a 4 hour incubation at 30०C, we purified the DNA from the reaction as described in the complete <html><a target="_blank" href="https://2019.igem.org/Team:TUDelft/Experiments#PURE">protocol: Expressing phi29 proteins in <I>PUREfrex</I> system.</a></html> The size of our construct was determined by DNA gel electrophoresis. As a control, we used samples with and without dNTPs. On our gel,  we observed that our linear construct (~2,3 kb) was successfully amplified, as shown in Figure 1. These results show that the phi29 replication machinery can be used for orthogonal DNA replication <I>in vitro</I>. We demonstrated that our origin-flanked linear construct (OriL-GFP-Kan-OriR) can be replicated <I>in vitro</I> by the phi29 minimal replication machinery.
  
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<div><center><ul>
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<li style="display: inline-block;"> [[File:T--TUDelft--ivttr.jpg|thumb|none|850px|<b>Figure 1:</b> Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+)  and without (-) dNTPs, amplified in vitro using the <I>PUREfrex</I> 2.0 system An intense band in the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the OriL-GFP-Kan-OriR in-vitro replication.]] </li>
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</ul></center></div>
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Successful replication of our linear construct has been shown on the agarose gel (Figure 1). A very intense band is visible at a size of 2.3 kb, corresponding to the size of OriL-GFP-Kan-OriR construct. This indicates that there has been successful <I>in vitro</I> amplification of our construct.
  
 
===References===
 
===References===
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<ul>
 
<ul>
 
<li>
 
<li>
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<a id="Lou2012" href="https://www.nature.com/articles/nbt.2401" target="_blank">
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<a id="Blanco1988" href="https://www.ncbi.nlm.nih.gov/pubmed/2498321" target="_blank">
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Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. <i>Nature Biotechnology</i>, 30(11), 1137–1142.</a>
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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>
 
</li>
 
</li>
 
<li>
 
<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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</ul>
 +
</html>

Latest revision as of 06:24, 14 December 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

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.

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 characterize our linear construct we wanted to demonstrate in vitro replication of our linear construct (OriL-GFP-Kan-OriR) and show that our construct can be propagated by the phi29 replication machinery. We used an in vitro transcription-translation system (PUREfrex®2.0) supplemented with dNTPs and purified auxiliary proteins: SSB and DSB, needed for an efficient DNA replication. We used 1nM as the starting DNA concentration of our OriL-GFP-Kan-OriR with a twofold excess of plasmid DNA encoding for both DNAP and TP. After a 4 hour incubation at 30०C, we purified the DNA from the reaction as described in the complete protocol: Expressing phi29 proteins in PUREfrex system. The size of our construct was determined by DNA gel electrophoresis. As a control, we used samples with and without dNTPs. On our gel, we observed that our linear construct (~2,3 kb) was successfully amplified, as shown in Figure 1. These results show that the phi29 replication machinery can be used for orthogonal DNA replication in vitro. We demonstrated that our origin-flanked linear construct (OriL-GFP-Kan-OriR) can be replicated in vitro by the phi29 minimal replication machinery.

  • Figure 1: Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+) and without (-) dNTPs, amplified in vitro using the PUREfrex 2.0 system An intense band in the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the OriL-GFP-Kan-OriR in-vitro replication.

Successful replication of our linear construct has been shown on the agarose gel (Figure 1). A very intense band is visible at a size of 2.3 kb, corresponding to the size of OriL-GFP-Kan-OriR construct. This indicates that there has been successful in vitro amplification of our construct.

References