<p>Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.<p>
<p>If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the <ahref="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria.</p>
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<p>Please see the <ahref="https://competition.igem.org/judging/medals">2022 Medals Page</a> for more information.</p>
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<h2><b>Engineering</b></h2>
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<h3><b>Design</b></h3>
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<p>At the design stage, we first did the preliminary research for our project. Through this, necessary enzymes and substances were identified, and expression vectors and genes were selected.<br>
The four enzymes we selected are as follows. Nitrite Reductase, Hydrazine Synthase, and Hydrazine Oxideneductase will be used for Nitrite (NO2-) and Ammonium (NH4+) removal.<br>
And superoxide dismutase enzymes will be used to perform H2O2 production.<br>
And we used this to construct parts. First, BBa_K4371000 (Anammox cluster: HZS + HZO + NIR) was created by clustering three enzymes used in Nitrite (NO2-) and Ammonium (NH4+) removal.<br>
It was inserted into pEXP5-NT, a specific cell free expression (CFE) vector.
Similarly, BBa_K38007 (SOD1 protein), a parts of Superoxide dismutase, an enzyme that conducts H2O2 production, was inserted into the expression vector pEXP5-NT.
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<h3><b>Build</b></h3>
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<p>In order to obtain the sequence found in the previous design stage, we ordered the sequence through Twist Bioscience. However, there was a problem that the sequence of the alpha subunit of the hydrazine synthase was longer than 1.8 kb. So we decided to get the gene by sequencing in the plasmid method.<br>
And we construct cell free expression system's experiment methods.</p>
<h3><b>Test</b></h3>
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<p>The T7 promoter shows the most active protein expression at 25°C.<br>
Figure. pT7 maximum Temperature (http://parts.igem.org/Help:Cell-free_chassis/Modelling#References)<br>
When checked through modeling, it can be seen that it is generated to the maximum at 25°C. </p>
<h3><b>Learn</b></h3>
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<p>In order to obtain more production, it is necessary to replace it with plasmid origin with a higher CN value or with a stronger RBS. Through this, more enzymes can be obtained and it can have advantages in producing filters.</p>
