<li> Summary: We created new way to characterize this biobrick which was originally designed and characterized by iGEM15_TU_Delft. We utilize NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) in our project to improve the characterization. We demonstrated the validity of the approach for measurement of biofilm composed by CsgA-His density of <i>E. coli</i> curli system and think highly of this characterization for its general application in other biofilm systems. Also, we utilized TEM to help us scrutinize the binding effect in microscopic world.</li>

<h3>>Improvement:</h3>

In order to prove the effect of binding between CsgA-Histag mutant and inorganic nanoparticles is distinct, we apply same amount of suspended CdSeS/ZnS QDs solution followed by the same procedure mentioned above. After 1h incubation, we used PBS washing 2 times. The picture verify our postulation: On the left, CsgA-Histag mutant were induced and its biofilm bind with QDS. CsgA biofilm without Histag cannot bind with QDs thus its red fluorescence is much weaker. </p>

As for biofilm characterization, transmission electron microscopy is frequently to be used to visualize the nanofiber network. However, TEM is not very efficient to visualize soft matter due to the less dense of elections produced on soft matter even after negative staining. Amazingly, after incubation with CdS nanorods , the biofilm areas are densely templated by better conductive materials such as CdS nanorods  and we can easily confirm the expression of biofilm.</p>

<b>Fig. 3</b>：Representative TEM images of biotemplated CdS nanorods on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with nanorods for 1h, nanomaterials are densely attached to biofilm.

<h4>

<li>Biobrick: <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>

<li> Summary: We optimized [FeFe] Hydrogenases originally from the bacterium <i>Clostridium acetobutylicum</i> (Original coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K535002">BBa_K535002</a>, designed by: iGEM11_UNAM-Genomics_ Mexico. Optimized coding sequence: hydA with SpyTag and Histag <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>) to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would produce more protein, theoretically. And optimization also improved the activity of [FeFe] Hydrogenases according to the experiment that we did.</li>

</ul></h4>

<h3>>Improvement:</h3>

<h4>Codon usage bias adjustment</h4>

<p>We analysed the Codon Adaptation Index (CAI) of the optimized coding sequence and the original one. And the distribution of codon usage frequency along the length of the gene sequence is increased from 0.33 to 0.97. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level.</p>

<figure align="center">

<img src="https://static.igem.org/mediawiki/2016/a/ab/SHTU_D1.png" width="70%">

<b>Fig. 4</b>：The distribution of codon usage frequency along the length of the gene sequence.

</figcaption>

</figure>

 

<p>We also compared the Frequency of Optimal Codons (FOP). The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. As we can see, the percentage of 91-100 increased largely, from 36 to 86, after the optimization.</p>

<figure align="center">

<img src="https://static.igem.org/mediawiki/2016/1/1c/SHTU_D2.png" width="70%">

<b>Fig. 5</b>：The percentage distribution of codons in computed codon quality groups.

</figcaption>

</figure>

 

<h4>What's more, we removed repeat sequences to break the Stem-Loop structures, which impact ribosomal binding and stability of mRNA.</h4>

<div class="col-lg-12">

<table align="center" border="0" cellpadding="0" cellspacing="0" class="table table-hover">

      <th><strong> </strong></th>

      <th><strong>Max Direct Repeat</strong></th>

      <th><strong>Max Inverted Repeat</strong></th>

      <th><strong>Max Dyad Repeat</strong></th>

      <td>After Optimization</a></td>

      <td>Size:15 Distance:3 Frequency:2</td>

      <td>None</td>

      <td>None</td>

      <td>Before Optimization</a></td>

      <td>Size:16 Distance:231 Frequency:2</td>

      <td>None</td>

      <td>Size: 13 Tm: 34.6 Start Positions: 680, 1357</td>

    </tr>

      <td colspan="12" align="center"><strong>Table 1: Removed repeat sequences information</strong></td>

    </tr>

      </tbody>

 

<h3>>Conclusion:</h3>

<p>A wide variety of factors regulate and influence gene expression levels, and after taking into consideration as many of them as possible, OptimumGene™ produced the single gene that can reach the highest possible level of expression.</p>

 

<p>In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in <em>E. coli</em> by upgrading the CAI from 0.33 to 0.97 . GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken.</p>

<figure align="center">

<img src="https://static.igem.org/mediawiki/2016/e/ea/SHTU_D3.png" width="70%">

<figcaption align="center">

<b>Fig. 6</b>：The protein alignment of new and old protein.

</figcaption>