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| − | Pbad-M13-Plac-GFP 3K3 K1139026 registry page
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| | __NOTOC__ | | __NOTOC__ |
| − | <partinfo>BBa_K1139026 short</partinfo> | + | <partinfo>BBa_K1139025 short</partinfo> |
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| − | M13 is a filamentous phage that infects only F+ strains of <i>E. coli</i>, which does not kill the host cell. This part is extracted from M13mp18 phage vector by PCR. It inclueds 11 ORFs, M13 origin, a packaging sequence and <i>lac</i> promoter. The promoter on the upstream of g2 (gene 2) is altered to <i>bad</i> promoter. A phage particle is formed only when the host cell receives arabinose signal because <i>g2p</i> (gene 2 protein) is an endonuclease needed for a plasmid to be replicated by M13 origin, and to be packaged into the phage particle.<br>
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| − | As a reporter, <i>GFP</i>. is inserted on the downstream of the <i>lac</i> promoter.<br>
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| − | We constructed a model system for inducible phage release by regulation of <i>g2p</i> expression. Genome DNA of this engineered phage, shown in Fig. 1, needs two functions. One is inducible expression of <i>g2p</i>. We thus designed to replace the promoter for <i>g2p</i> with <i>bad</i> promoter. Note that we used arabinose in this model experiment. The other is maintenance of the genome DNA in the absence of <i>g2p</i> expression. We combined M13 genome double stranded DNA with pSB3K3 backbone.<br>
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| − | [[Image:titech2013_parts_K1139021_main_Fig1.png|thumb|center|300px|<b>Fig. 1.</b> Design of phage DNA for inducible phage release pSB3K3 backbone is required for maintenance of the DNA without inducer.]]
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| − | Firstly, we confirmed that M13 genome with two modifications related to our design kept plaque forming activity. One is replacement of the promoter for <i>g2p</i> with a constitutive promoter, PLacI<sup>q</sup> (<partinfo>BBa_I14032</partinfo>). The other is accommodation of pSB3K3 backbone. Even though the plasmid has two different types of replication origins, M13 origin and pSB3 origin, this plasmid (<partinfo>BBa_K1139020</partinfo>) formed plaque. In contrast, construction intermediates without a promoter upstream of <i>g2p</i> coding sequence (Promoterless-M13 + Plac: <partinfo>BBa_K1139018</partinfo>, Promoterless-M13 + Plac-<i>GFP</i>: <partinfo>BBa_K1139022</partinfo>) could not form plaque.<br>
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| − | We then confirmed that replacement of the <i>g2p</i> promoter with <i>bad</i> promoter accomplished inducible phage release. For a plaque forming assay, we used a plasmid with <i>bad</i> promoter upstream of <i>g2p</i> coding sequence (Pbad-M13-Plac-<i>GFP</i>: <partinfo>BBa_K1139026</partinfo>). Besides, as lawn, we used JM109 (F<sup>+</sup> strain) which has a plasmid that is araC<sup>+</sup>. Also, to make a concentration gradient of the inducer (arabinose), we put a piece of filter paper on which dropped the inducer solution. <br>
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| − | [[Image:titech2013_parts_K1139021_main_Fig2.png|thumb|center|300px|<b>Fig. 2.</b> We used a new biobrick part (<partinfo>BBa_K1139026</partinfo>), for the assay. The expression of <i>g2p</i> is activated by the induction, resulting in release of phage particles. We used the phage particles for the plaque forming assay. (To make a concentration gradient, we put a piece of filter paper on the plate and dripped inducer on the piece of filter paper.)]]
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| − | The result shows that the plaques are formed only when the inducer exists in the medium (Fig. 3). In addition, the distribution of the plaques has an optimum value, depending on the distance from the piece of filter paper which has soaked up the inducer (Fig. 4). <br>
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| − | In the neighborhood of the piece of filter paper, the expression of <i>g2p</i> is so activated that the phage particles cannot be produced efficiently, because the production of the coat proteins largely exceeds that of the single stranded DNA, and vice versa.<br>
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| − | This result shows that the inducible release of M13 phage is realized; our plan was achieved.<br>
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| − | [[Image: |thumb|center|300px|<b>Fig. 3.</b> Conclusion that AHL included filter paper induced phage release]]
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| − | [[Image: |thumb|center|300px|<b>Fig. 4.</b> The distribution histogram of the plaques]]
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| − | For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/Inducible_Plaque_Forming_Assay our work in Tokyo_Tech 2013 wiki].
| + | M13 is a filamentous phage that infects only F+ strains of <i>E. coli</i>, which does not kill the host cell. <br> |
| | + | This part is derived from M13mp18 phage vector(NEB), amplified by PCR. It includes 11 ORFs, M13 origin and a packaging sequence. The promoter upstream of <i>g2p</i> (gene 2 protein) is altered to <i>bad</i> promoter. A phage particle is formed only when the host cell receives arabinose because <i>g2p</i> is an endonuclease needed for a plasmid to be replicated by M13 origin, and to be packaged into the phage particle.<br> |
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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
| − | <partinfo>BBa_K1139026 SequenceAndFeatures</partinfo> | + | <partinfo>BBa_K1139025 SequenceAndFeatures</partinfo> |
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| | <!-- Uncomment this to enable Functional Parameter display | | <!-- Uncomment this to enable Functional Parameter display |
| | ===Functional Parameters=== | | ===Functional Parameters=== |
| − | <partinfo>BBa_K1139026 parameters</partinfo> | + | <partinfo>BBa_K1139025 parameters</partinfo> |
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