Difference between revisions of "Part:BBa K3699007"

 
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<html>
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    <h3 id="degradation0301">Design </h3>
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      <p>
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            The “tail” protein encodes the putative phage tail sheath protein of <i>Microcystis virus</i> Ma-LMM01.
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            (Protein ID: BAF36182.1)</p>
 +
        <p>We have made a base substitution at position 747 (T747G), which converted the Tyrosine codon to a stop codon
 +
            and lead to the early termination of protein translation. In order to facilitate plasmid conctruction, we
 +
            added BamHI and SacI cutting sites at both ends of the gene.</p>
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        <div class="imgBox">
 +
            <img style="margin: 20px auto;" src="https://2020.igem.org/wiki/images/5/5c/T--BUCT--eng10.png" alt="chart" />
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        </div>
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        <div class="imgBox">
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            <img style="margin: 20px auto;" src="https://2020.igem.org/wiki/images/2/24/T--BUCT--eng8.png"alt="chart" />
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        </div>
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        <p><b>Figure 1. sequence of the tail protein.</b> We made a base substitution at position 747 (T747G), converting TAT
 +
            to TAG.</p>
 +
        <p>We speculate that the mutated phage tail sheath protein will not be fully expressed, but a truncated protein.
 +
        </p>
  
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__NOTOC__
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    <h3 id="degradation0301">Construction </h3>
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<partinfo>BBa_K3699007 short</partinfo>
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        <p>
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            Plasmid pKMV-Tail was synthesized by BGI Tech. On this basis, we constructed the plasmid pET28a-Tail by
 +
            digesting pKMV-tail and pET28a with BamHI/SacI enzyme digestion and ligation. Plasmid pET28a, which contains
 +
            T7 promotor, is an excellent expression vector in <i>E. coli</i> BL21 (DE3) with IPTG addition. The tail
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            protein gene will be highly expressed.</p>
 +
        <div class="imgBox">
 +
            <img style="margin: 20px auto;" src="https://2020.igem.org/wiki/images/9/94/T--BUCT--eng5.png
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        " alt="chart" />
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        </div>
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        <p><b>Figure 2. Skeleton map of pET28a-Tail.</b></p>
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        <div class="imgBox">
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            <img style="margin: 20px auto;" src="
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            https://2020.igem.org/wiki/images/2/20/T--BUCT--eng9.png
 +
        " alt="chart" />
 +
        </div>
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        <p><b>Figure 3. Construction of pET28a-Tail.</b> BamHI-SacI double enzyme digestion of pKMV-tail and pET28a to
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            construct plasmid pET28a-Tail. T7 promoter was introduced to facilitate expression in <i>E. coli</i>.</p>
  
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the phage tail sheath protein, aiming to detect the orthogonal translation system
+
       
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    <h3 id="degradation0301">Testing </h3>
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        <p>
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            We demonstrated the success of plasmid construction by enzymatic digestion.</p>
 +
        <div class="imgBox">
 +
            <img style="margin: 20px auto;" src="
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        https://2020.igem.org/wiki/images/1/16/T--BUCT--eng4.png
 +
        " alt="chart" />
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        </div>
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        <p><b>Figure 4. DNA gel electrophoresis for pET28a-Tail digensted with BamHI-SacI (2.3kb + 5.3kb).</b></p>
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    <h3 id="degradation0301">Expression of the truncated Tail protein </h3>
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 +
        <p>
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            We transferred pET28a-Tail into <i>E. coli</i> BL21 (DE3). As expected, it should express a truncated
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            protein of 29.7 kDa.</p>
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        <div class="imgBox">
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            <img style="margin: 20px auto;" src="
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        https://2020.igem.org/wiki/images/b/bd/T--BUCT--eng6.png
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        " alt="chart" />
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        </div>
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        <p><b>Figure 5. The expected truncated tail protein sequence.</b></p>
  
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<!-- Add more about the biology of this part here
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    <h3 id="degradation0301">Result </h3>
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===Usage and Biology===
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        <div class="imgBox">
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            <img style="margin: 20px auto;" src="
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<!-- -->
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            https://2020.igem.org/wiki/images/3/3d/T--BUCT--eng11.png
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<span class='h3bb'>Sequence and Features</span>
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        " alt="chart" />
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<partinfo>BBa_K3699007 SequenceAndFeatures</partinfo>
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        </div>
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        <p><b>Figure 6. SDS page of BL21 (DE3) harboring plasmid pET28a-Tail.</b></p>
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        <p>From left to right:</p>
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<!-- Uncomment this to enable Functional Parameter display
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        <p>1. Bacterial lysis, no IPTG; 2. Bacterial lysis, IPTG induction;</p>
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===Functional Parameters===
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        <p>3. Bacteria cell pellets, no IPTG; 4. Bacterial cell pellets, IPTG induction; M protein marker</p>
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<partinfo>BBa_K3699007 parameters</partinfo>
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        <p>
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<!-- -->
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            <b>The introduction of stop codon caused the early termination of protein translation.</b> The recombinant <i>E.
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                coli</i> expressed a truncated phase tail shear protein as expected. In other words, <b>if the stop codon
 +
            was introduced into the cyanophage genome, it will become a virus that can not release or infect
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            cyanobacteria, thus preventing the escape of recombinant phage.</b></p>
 +
 
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    <h3 id="degradation0301">Reference </h3>
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        <p>[1] Daichi M , Shigeko K , Yoshihiko S , et al. Transcriptome Analysis of a Bloom-Forming Cyanobacterium
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            Microcystis aeruginosa during Ma-LMM01 Phage Infection[J]. Frontiers in Microbiology, 2018, 9:2-.
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        </p>
 +
        <p>[2] Mukai T , Kobayashi T , Hino N , et al. Adding l-lysine derivatives to the genetic code of mammalian
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            cells with engineered pyrrolysyl-tRNA synthetases[J]. Biochemical & Biophysical Research Communications,
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            2008, 371(4):818-822.
 +
        </p>
 +
       
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</html>

Revision as of 23:53, 27 October 2020

Design

The “tail” protein encodes the putative phage tail sheath protein of Microcystis virus Ma-LMM01. (Protein ID: BAF36182.1)

We have made a base substitution at position 747 (T747G), which converted the Tyrosine codon to a stop codon and lead to the early termination of protein translation. In order to facilitate plasmid conctruction, we added BamHI and SacI cutting sites at both ends of the gene.

chart
chart

Figure 1. sequence of the tail protein. We made a base substitution at position 747 (T747G), converting TAT to TAG.

We speculate that the mutated phage tail sheath protein will not be fully expressed, but a truncated protein.

Construction

Plasmid pKMV-Tail was synthesized by BGI Tech. On this basis, we constructed the plasmid pET28a-Tail by digesting pKMV-tail and pET28a with BamHI/SacI enzyme digestion and ligation. Plasmid pET28a, which contains T7 promotor, is an excellent expression vector in E. coli BL21 (DE3) with IPTG addition. The tail protein gene will be highly expressed.

chart

Figure 2. Skeleton map of pET28a-Tail.

chart

Figure 3. Construction of pET28a-Tail. BamHI-SacI double enzyme digestion of pKMV-tail and pET28a to construct plasmid pET28a-Tail. T7 promoter was introduced to facilitate expression in E. coli.

Testing

We demonstrated the success of plasmid construction by enzymatic digestion.

chart

Figure 4. DNA gel electrophoresis for pET28a-Tail digensted with BamHI-SacI (2.3kb + 5.3kb).

Expression of the truncated Tail protein

We transferred pET28a-Tail into E. coli BL21 (DE3). As expected, it should express a truncated protein of 29.7 kDa.

chart

Figure 5. The expected truncated tail protein sequence.

Result

chart

Figure 6. SDS page of BL21 (DE3) harboring plasmid pET28a-Tail.

From left to right:

1. Bacterial lysis, no IPTG; 2. Bacterial lysis, IPTG induction;

3. Bacteria cell pellets, no IPTG; 4. Bacterial cell pellets, IPTG induction; M protein marker

The introduction of stop codon caused the early termination of protein translation. The recombinant E. coli expressed a truncated phase tail shear protein as expected. In other words, if the stop codon was introduced into the cyanophage genome, it will become a virus that can not release or infect cyanobacteria, thus preventing the escape of recombinant phage.

Reference

[1] Daichi M , Shigeko K , Yoshihiko S , et al. Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection[J]. Frontiers in Microbiology, 2018, 9:2-.

[2] Mukai T , Kobayashi T , Hino N , et al. Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases[J]. Biochemical & Biophysical Research Communications, 2008, 371(4):818-822.