Difference between revisions of "Part:BBa K2918034"

 
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<partinfo>BBa_K2918034 short</partinfo>
 
<partinfo>BBa_K2918034 short</partinfo>
  
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Broad host range promoter expected to work in <i> E.coli <i/> and <i> B.subtilis </i>.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2918034 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2918034 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
 +
The broad host range promoter has been designed by combining promoter regions of <i>E.coli</i> , <i> B.subtilis </i> and <i> S.cerevisiae </i>. The promoter is based on the Pmin minimal promoter of S.cerevisiae.  It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in <i> E.coli</i> and <i>B.subtilis </i>. Therefore, to make the Pmin promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the Pmin promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′.
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 +
===Strain Construction===
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Strain construction:
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Aim: To clone the promoter in a level 0 MoClo backbone <html> (<a target=”_blank” href=”https://www.addgene.org/47984/”>pICH41233</a>) </html>.
 +
Procedure: The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible promoter overhangs. 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).
 +
Click <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank">here</a> </html> for the protocol.
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 +
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<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>
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<html>
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    <style>
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        #tabletu {
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            background-color: transparent;
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            border-collapse: collapse;
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            width:80%;
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        }
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        #tabletu td, th {
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            border: 1px solid #000000;
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            padding: 8px;
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        }
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        #tabletu th {
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            padding: 8px;
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            text-align: left;
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            border: 1px solid #000000; 
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            background-color: rgba(0,110,167,1);
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            color: white;
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        }
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    </style>
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    <body>
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        <b>Table 1:</b> Overview of different level in MoClo
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        <table id="tabletu">
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            <tr>
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                <th>Level
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                </th>
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                <th>Basic/Composite
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                </th>
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                <th>
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                    Type</th>
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                <th>Enzyme</th>
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            </tr>
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            <tr>
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                <td>
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                    Level 0
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                </td>
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                <td>Basic</td>
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                <td>Promoters, 5’ UTR, CDS and terminators</td>
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                <td>BpiI</td>
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            </tr>
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            <tr>        <td>Level 1</td>
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                <td>Composite</td>
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                <td>Transcriptional units</td>
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                <td>BsaI</td>
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            </tr>
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            <tr>
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                <td>
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                    Level 2/M/P</td>
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                <td>Composite</td>
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                <td>Multiple transcriptional units</td>
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                <td>BpiI</td>
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            </tr>
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        </table>
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    </body>
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</html>
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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.
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<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;
 +
        }
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 +
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    </style>
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 +
    <body>
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        <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
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        <table id="tabletu">
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            <tr>
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                <th>Basic Part
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                </th>
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                <th>Sequence 5' End
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                </th>
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                <th>
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                    Sequence 3' End</th>
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                <th>Level 0 backbone</th>
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            </tr>
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            <tr>
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                <td>
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                    Promoter
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                </td>
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                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GGAG</span></td>
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                <td><span style="color:dodgerblue">TACT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
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                <td>pICH41233</td>
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            </tr>
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            <tr>        <td>5’ UTR</td>
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                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">TACT</span></td>
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                <td><span style="color:dodgerblue">AATG</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
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                <td>pICH41246</td>
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            </tr>
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            <tr>
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                <td>
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                    CDS</td>
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                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">AATG</span></td>
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                <td><span style="color:dodgerblue">GCTT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
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                <td>pICH41308</td>
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            </tr>
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            <tr>
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                <td>
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                    Terminator</td>
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                <td>NNNN <span style="color:limegreen">GAAGAC</span> NN <span style="color:dodgerblue">GCTT</span></td>
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                <td><span style="color:dodgerblue">CGCT</span> NN <span style="color:limegreen">GTCTTC</span> NNNN</td>
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                <td>pICH41276</td>
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            </tr>
 +
 +
 +
        </table>
 +
 +
    </body>
 +
</html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2918034 parameters</partinfo>
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<partinfo>BBa_K2918005 parameters</partinfo>
 
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Revision as of 13:51, 6 October 2019

Φ29 DNA polymerase (DNAP/p2)

Broad host range promoter expected to work in E.coli <i/> and <i> B.subtilis .

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1662
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The broad host range promoter has been designed by combining promoter regions of E.coli , B.subtilis and S.cerevisiae . The promoter is based on the Pmin minimal promoter of S.cerevisiae. It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in E.coli and B.subtilis . Therefore, to make the Pmin promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the Pmin promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′.

Strain Construction

Strain construction: Aim: To clone the promoter in a level 0 MoClo backbone (pICH41233) . Procedure: The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible promoter overhangs. 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). Click here for the protocol.


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