Difference between revisions of "Team:Gifu/Improve"

 
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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<h1>Improvement plan</h1><hr>
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<p>*Caution: We did not submit the part to iGEM HQ this year. Since linear DNA is workable, we evaluate how much important the enhancer is for  PURE system and T7 RNA polymerase. We will submit this part in the competition of 2019. Thus this part is not met the gold criteria.</p>
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<p>As proof of improvement of previous composite part of iGEM, <a href="http://parts.igem.org/Part:BBa_K1491033">BBa_K1491033</a> was provided by iGEM HQ. Typical alignment of T7 promoter is used in this part. However, in PURE system, the enhancer of the stem-loop structure need to be located in the downstream of T7 promoter to make an expression of protein intense. Thus we also design the part (Seq.2) below to show the improvement and this may contribute to the development of synthetic biology and iGEM competition. In Fig.1 we showed the detail of T7 promoter we used this year. Translation enhancer upstream of the SD sequence of mRNA, enhance the biosynthesis of protein. The enhancer may contribute to a direct interaction with ribosome protein S1. Fig.1 shows the detail of the enhancer.</p>
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<p>*Caution: BBa_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>. In Seq.2 it has s stop codon.</p>
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      <img src="https://static.igem.org/mediawiki/2018/f/f7/T--Gifu--improve3.png" width="600px"><br>
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      <center><legend><b>Seq.2 T7p--SD-RFP-T7t</b></legend></center>
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      <img src="https://static.igem.org/mediawiki/2018/0/09/T--Gifu--promoter.png" width="600px"><br>
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      <img src="https://static.igem.org/mediawiki/2018/6/6e/T--Gifu--BASE.png" width="600px"><br>
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<center><legend><b>Fig.1 Explanation of enhancer of T7 promoter</b></legend></center>
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<h1>Improvement Result</h1><hr>
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<p>As a result of the experiment, we can see the clear fluorescence in our new part, but not in BBa_K1491033.</p>
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  <img src="https://static.igem.org/mediawiki/2018/7/7d/T--Gifu--UV_PURE.jpg" width="700px"><br>
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  <center> <legend><b>Fig.4 Expression in PURE system under UV</b><p>P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA</p>
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<p>In addition, we can see the apparent difference in SDS-PAGE.</p>
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<img src="https://static.igem.org/mediawiki/2018/7/76/T--Gifu--SDSPAGE.jpg" height="330px">
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<img src="https://static.igem.org/mediawiki/2018/6/6f/T--Gifu--SDSPAGE-LANE.png" height="330px">
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  <legend><b>Fig.4 SDSPAGE</b></center>
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Latest revision as of 03:14, 18 October 2018

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Improvement plan

*Caution: We did not submit the part to iGEM HQ this year. Since linear DNA is workable, we evaluate how much important the enhancer is for PURE system and T7 RNA polymerase. We will submit this part in the competition of 2019. Thus this part is not met the gold criteria.

As proof of improvement of previous composite part of iGEM, BBa_K1491033 was provided by iGEM HQ. Typical alignment of T7 promoter is used in this part. However, in PURE system, the enhancer of the stem-loop structure need to be located in the downstream of T7 promoter to make an expression of protein intense. Thus we also design the part (Seq.2) below to show the improvement and this may contribute to the development of synthetic biology and iGEM competition. In Fig.1 we showed the detail of T7 promoter we used this year. Translation enhancer upstream of the SD sequence of mRNA, enhance the biosynthesis of protein. The enhancer may contribute to a direct interaction with ribosome protein S1. Fig.1 shows the detail of the enhancer.

*Caution: BBa_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as BBa_E1010. In Seq.2 it has s stop codon.


Seq.2 T7p--SD-RFP-T7t


Fig.1 Explanation of enhancer of T7 promoter

Improvement Result

As a result of the experiment, we can see the clear fluorescence in our new part, but not in BBa_K1491033.


Fig.4 Expression in PURE system under UV

P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA

In addition, we can see the apparent difference in SDS-PAGE.

Fig.4 SDSPAGE