Our system provided a way to regulate microbiota to an ideal balance . Since we focused our model on the excess PSB in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the Bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed BioBricks that contain sequences of these peptides. All the experiments we performed with BioBricks are mentioned below.
Difference between revisions of "Team:NCTU Formosa/Wet Lab"
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<svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg> | <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg> | ||
| − | Figure 1 | + | Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD |
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<div class="explanation"> | <div class="explanation"> | ||
<svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg> | <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg> | ||
| − | Figure 2 | + | Figure 2: Procedure of intein-mediated protein splicing |
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<div class="title_1"><p>1. Protein expression</p></div> | <div class="title_1"><p>1. Protein expression</p></div> | ||
<div class="title_2"><p>Cloning:</p></div> | <div class="title_2"><p>Cloning:</p></div> | ||
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<p>We cloned the insert gene into pSB1C3 backbone and transform into <i>E. coli</i> DH5α. <a href="https://2018.igem.org/Team:NCTU_Formosa/Parts parts">These</a> have been submitted to iGEM.</p> | <p>We cloned the insert gene into pSB1C3 backbone and transform into <i>E. coli</i> DH5α. <a href="https://2018.igem.org/Team:NCTU_Formosa/Parts parts">These</a> have been submitted to iGEM.</p> | ||
</div> | </div> | ||
<div class="title_2"><p>Expression:</p></div> | <div class="title_2"><p>Expression:</p></div> | ||
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<p>We expressed the proteins by <i>E. coli</i> ER2566 and <i>E. coli</i> BL21 Rosetta-Gami.</p> | <p>We expressed the proteins by <i>E. coli</i> ER2566 and <i>E. coli</i> BL21 Rosetta-Gami.</p> | ||
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<div class="title_2"><p>SDS-PAGE:</p></div> | <div class="title_2"><p>SDS-PAGE:</p></div> | ||
| − | <div class=" | + | <div class="textnew"> |
<p>We checked the expression result through SDS-PAGE.</p> | <p>We checked the expression result through SDS-PAGE.</p> | ||
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Revision as of 16:49, 30 September 2018
Experiment design
BioBrick
The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a intein-CBD (chitin binding domain) tag behind bacteriocin to better purify our peptide.
Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD
Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides. Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.
Figure 2: Procedure of intein-mediated protein splicing
1. Protein expression
Cloning:
We cloned the insert gene into pSB1C3 backbone and transform into E. coli DH5α. These have been submitted to iGEM.
Expression:
We expressed the proteins by E. coli ER2566 and E. coli BL21 Rosetta-Gami.
SDS-PAGE:
We checked the expression result through SDS-PAGE.
2. MIC test
Inhibition zone:
Minimum Inhibitory Concentration Broth Microdilution:
3. Growth curve experiment
4. Soil experiment
