Difference between revisions of "Part:BBa K2918029"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2918029 short</partinfo>
 
<partinfo>BBa_K2918029 short</partinfo>
  
 +
Linear plasmid for replication by Φ29 replication machinery.
  
−
 
−
 
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2918029 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2918029 SequenceAndFeatures</partinfo>
 +
The part has been confirmed by sequencing and there are no mutations.
  
 +
===Overview===
  
−
<!-- Uncomment this to enable Functional Parameter display
+
<p>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 replication, greatly simplifies the design of replication systems. The Φ29 replication begins at origins of replication (OriR and OriL) on two sides of a linear protein primed linear DNA <html><a href="#Nies2018">(Nies et al, 2018)</a></html>.The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2),terminal protein [https://parts.igem.org/Part:BBa_K2918001 (TP/p3)],single stranded binding protein [https://parts.igem.org/Part:BBa_K2918002 (SSB/p5)] and double stranded binding protein [https://parts.igem.org/Part:BBa_K2918003 (DSB/p6)].
−
===Functional Parameters===
+
The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication <html><a href="#Nies2018">(Nies et al, 2018)</a></html>.The Φ29 DNA polymerase is a single subunit protein known to have high processivity, it has been shown to synthesize upto 70 kilobase pairs of DNA <html><a href="#Blanco1988">(Blanco et al, 1988)</a></html>. The double stranded DNA binding proteins [https://parts.igem.org/Part:BBa_K2918003 (DSB/p6)] aid in the process of replication and binds 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>.</p>
−
<partinfo>BBa_K2918029 parameters</partinfo>
+
 
−
<!-- -->
+
===Strain Construction===
 +
The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors [http://www.addgene.org/47998/pICH47732] and [http://www.addgene.org/47998/pICH47742] by BpiI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BsaI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector [http://www.addgene.org/47998/pICH8031] and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.
 +
 
 +
===Modular Cloning===
 +
Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening).
 +
For the protocol, you can find it <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html>
 +
 
 +
 
 +
<b>Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a>.</html></b>
 +
 
 +
 
 +
<html>
 +
    <style>
 +
 
 +
        #tabletu {
 +
            background-color: transparent;
 +
            border-collapse: collapse;
 +
            width:80%;
 +
        }
 +
 
 +
        #tabletu td, th {
 +
            border: 1px solid #000000;
 +
            padding: 8px;
 +
        }
 +
 
 +
        #tabletu th {
 +
            padding: 8px;
 +
            text-align: left;
 +
            border: 1px solid #000000; 
 +
            background-color: rgba(0,110,167,1);
 +
            color: white;
 +
        }
 +
 
 +
    </style>
 +
 
 +
    <body>
 +
        <b>Table 1:</b> Overview of different level in MoClo
 +
        <table id="tabletu">
 +
            <tr>
 +
                <th>Level
 +
                </th>
 +
                <th>Basic/Composite
 +
                </th>
 +
                <th>
 +
                    Type</th>
 +
                <th>Enzyme</th>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    Level 0
 +
                </td>
 +
                <td>Basic</td>
 +
                <td>Promoters, 5’ UTR, CDS and terminators</td>
 +
                <td>BpiI</td>
 +
 
 +
            </tr>
 +
            <tr>        <td>Level 1</td>
 +
                <td>Composite</td>
 +
                <td>Transcriptional units</td>
 +
                <td>BsaI</td>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    Level 2/M/P</td>
 +
                <td>Composite</td>
 +
                <td>Multiple transcriptional units</td>
 +
                <td>BpiI</td>
 +
            </tr>
 +
 
 +
 
 +
        </table>
 +
 
 +
 
 +
    </body>
 +
</html>
 +
 
 +
For synthesizing basic parts, the part of interest should be flanked by a <span style="color:limegreen">BpiI site</span> and its <span style="color:dodgerblue">specific type overhang</span>. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.
 +
 
 +
 
 +
 
 +
<html>
 +
    <style>
 +
 
 +
        #tabletu {
 +
            background-color: transparent;
 +
            border-collapse: collapse;
 +
            width:100%;
 +
        }
 +
 
 +
        #tabletu td, th {
 +
            border: 1px solid #000000;
 +
            padding: 8px;
 +
        }
 +
 
 +
        #tabletu th {
 +
            padding: 8px;
 +
            text-align: left;
 +
            border: 1px solid #000000; 
 +
            background-color: rgba(0,110,167,1);
 +
            color: white;
 +
        }
 +
 
 +
 
 +
    </style>
 +
 
 +
    <body>
 +
        <b>Table 2:</b> Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts
 +
        <table id="tabletu">
 +
            <tr>
 +
                <th>Basic Part
 +
                </th>
 +
                <th>Sequence 5' End
 +
                </th>
 +
                <th>
 +
                    Sequence 3' End</th>
 +
                <th>Level 0 backbone</th>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    Promoter
 +
                </td>
 +
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GGAG</span></td>
 +
                <td><span style="color:dodgerblue">TACT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
 +
                <td>pICH41233</td>
 +
 
 +
            </tr>
 +
            <tr>        <td>5’ UTR</td>
 +
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">TACT</span></td>
 +
                <td><span style="color:dodgerblue">AATG</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
 +
                <td>pICH41246</td>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    CDS</td>
 +
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">AATG</span></td>
 +
                <td><span style="color:dodgerblue">GCTT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
 +
                <td>pICH41308</td>
 +
            </tr>
 +
            <tr>
 +
                <td>
 +
                    Terminator</td>
 +
                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GCTT</span></td>
 +
                <td><span style="color:dodgerblue">CGCT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
 +
                <td>pICH41276</td>
 +
            </tr>
 +
 
 +
 
 +
        </table>
 +
 
 +
 
 +
    </body>
 +
</html>
 +
 
 +
===Characterization===
 +
 
 +
 
 +
===References===
 +
<html>
 +
<ul>
 +
<li>
 +
<a id="Nies2018" href="https://www.nature.com/articles/s41598-018-31585-1" target="_blank">
 +
Nies, P. Van, Westerlaken, I., Blanken, D., Salas, M., Mencía, M., & Danelon, C. (n.d.). Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. <i>Nature Communications</i>, (2018), 1–12. https://doi.org/10.1038/s41467-018-03926-1 </a>
 +
</li>
 +
<li>
 +
<a id="Blanco1988" href="https://www.ncbi.nlm.nih.gov/pubmed/2498321" target="_blank">
 +
Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.</a>
 +
</li>
 +
</ul>
 +
 
 +
</html>

Revision as of 13:53, 18 October 2019

OriL-GFP-Kan-OriR

Linear plasmid for replication by Φ29 replication machinery.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 664
    Illegal EcoRI site found at 2056
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 664
    Illegal EcoRI site found at 2056
    Illegal NheI site found at 956
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 664
    Illegal EcoRI site found at 2056
    Illegal BamHI site found at 873
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 664
    Illegal EcoRI site found at 2056
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 664
    Illegal EcoRI site found at 2056
  • 1000
    COMPATIBLE WITH RFC[1000]

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

Overview

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 replication, greatly simplifies the design of replication systems. The Φ29 replication begins at origins of replication (OriR and OriL) on two sides of a linear protein primed linear DNA (Nies et al, 2018).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 (Nies et al, 2018).The Φ29 DNA polymerase is a single subunit protein known to have high processivity, it has been shown to synthesize upto 70 kilobase pairs of DNA (Blanco et al, 1988). The double stranded DNA binding proteins (DSB/p6) aid in the process of replication and binds 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).

Strain Construction

The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors [http://www.addgene.org/47998/pICH47732] and [http://www.addgene.org/47998/pICH47742] by BpiI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BsaI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector [http://www.addgene.org/47998/pICH8031] and the construct was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). For the protocol, you can find it here.


Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found here.


Table 1: Overview of different level in MoClo

Level Basic/Composite Type Enzyme
Level 0 Basic Promoters, 5’ UTR, CDS and terminators BpiI
Level 1 Composite Transcriptional units BsaI
Level 2/M/P Composite Multiple transcriptional units BpiI

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.


Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Basic Part Sequence 5' End Sequence 3' End Level 0 backbone
Promoter NNNN GAAGAC NN GGAG TACT NN GTCTTC NNNN pICH41233
5’ UTR NNNN GAAGAC NN TACT AATG NN GTCTTC NNNN pICH41246
CDS NNNN GAAGAC NN AATG GCTT NN GTCTTC NNNN pICH41308
Terminator NNNN GAAGAC NN GCTT CGCT NN GTCTTC NNNN pICH41276

Characterization

References