Difference between revisions of "Team:Linkoping Sweden/Improve"

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<a href="https://2018.igem.org/Team:Linkoping_Sweden">
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              <a href="/Team:Linkoping_Sweden/Demonstrate">Demonstrate</a>
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              <a href="/Team:Linkoping_Sweden/Improve">Improve</a>
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<section class="superproject">
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<h1>Improve</h1>
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<h1>
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Improvement of Liu iGEM 2017s part BBa_K2474000
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With the use of site-directed mutagenesis the goal was to improve the part of last years LiU iGEM team biobrick BBa_K2474000 and make it to a reporter system which could be more widely used. The improved biobrick made this year (BBa_K2671000) in contrast to BBa_K2474000 could also be used to display how aggregation prone proteins affect fused reporters. This was done by removing amyloid-beta 1-42 via adding a restiction site (SpeI) and removing unnecessary bases as shown in figure 1. By doing this the fluorescent protein mNeonGreen (mNG) can be used as an efficient reporter gene with a different protein, instead of amyloid-beta 1-42. The promoter of this system is AraC/pBAD system [1]. <br><br>The two additions were made with site-directed mutagenesis. Firstly the stop codon was added and sequenced, which was later used as the new template for the mutation to add the SpeI-site. As seen in figure 1, SpeI was used to cleave off the unnecessary bases from the plasmid, and later ligated it back together without the amyloid-beta 1-42 fragment. </br>
  
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<img src="https://static.igem.org/mediawiki/2018/d/d1/T--Linkoping_Sweden--improve.jpg"></img>
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<i>Figure 1. A illutration of the steps made.</i>
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<h3>
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Results
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The results for our improved part can be seen in the figure below. The reason we chose to improve BBa_K2474000, as previously mentioned,  was mainly to create a functional reporter system. However, this new part (BBa_K2671000) is able to illustrate how the loss of  an aggregation prone fusion tag increase flouresence intensity. From Figure 2 it is visible that the new version without amyloid-beta 1-42 fused has an increased intensity when compared to the unmutated part.
  
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The two parts were grown in LB-medium, 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in Figure 2
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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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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<img src="https://static.igem.org/mediawiki/parts/a/a3/T--Linkoping_Sweden--improved.jpeg"></img>
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<i>Figure 2. From left to right: Reference with only water, BBa_K2671000 (new improved mutated part made by LiU iGEM 2018) and BBa_K2474000 (unmutated part made by LiU iGEM 2017).</i>
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Sequencing
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Seen below in Figure 3 is the chromatograms from sequencing. The results shows that the mutation GCC --> TTA was successful. The length of the sequence also suggest that amyloid-beta 1-42 was removed. Both parts was sequenced using the VR primer, and ~170 bases shorter fits completely with the length of the removed fragment. The full sequencing files including chromatograms can be found at <a target="_blank" style="color:blue" href="http://parts.igem.org/Part:BBa_K2671000">BBa_K2671000</a>.
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<img src="https://static.igem.org/mediawiki/parts/c/c2/T--Linkoping_Sweden--chromatogram.png"> </H4>
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<i>Figure 3. The chromatograms from sequencing. Left is BBa_K2671000 (mutated part) and right is BBa_K2474000 (template).</i>
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References
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<H4>[1] Part:BBa K2474000 - parts.igem.org [Internet]. [cited 2018 Oct 18]. Available from: http://parts.igem.org/Part:BBa_K2474000 </H4>
  
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Latest revision as of 23:29, 17 October 2018

LiU iGEM

Improve

Improvement of Liu iGEM 2017s part BBa_K2474000

With the use of site-directed mutagenesis the goal was to improve the part of last years LiU iGEM team biobrick BBa_K2474000 and make it to a reporter system which could be more widely used. The improved biobrick made this year (BBa_K2671000) in contrast to BBa_K2474000 could also be used to display how aggregation prone proteins affect fused reporters. This was done by removing amyloid-beta 1-42 via adding a restiction site (SpeI) and removing unnecessary bases as shown in figure 1. By doing this the fluorescent protein mNeonGreen (mNG) can be used as an efficient reporter gene with a different protein, instead of amyloid-beta 1-42. The promoter of this system is AraC/pBAD system [1].

The two additions were made with site-directed mutagenesis. Firstly the stop codon was added and sequenced, which was later used as the new template for the mutation to add the SpeI-site. As seen in figure 1, SpeI was used to cleave off the unnecessary bases from the plasmid, and later ligated it back together without the amyloid-beta 1-42 fragment.

Figure 1. A illutration of the steps made.

Results

The results for our improved part can be seen in the figure below. The reason we chose to improve BBa_K2474000, as previously mentioned, was mainly to create a functional reporter system. However, this new part (BBa_K2671000) is able to illustrate how the loss of an aggregation prone fusion tag increase flouresence intensity. From Figure 2 it is visible that the new version without amyloid-beta 1-42 fused has an increased intensity when compared to the unmutated part. The two parts were grown in LB-medium, 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in Figure 2

Figure 2. From left to right: Reference with only water, BBa_K2671000 (new improved mutated part made by LiU iGEM 2018) and BBa_K2474000 (unmutated part made by LiU iGEM 2017).



Sequencing

Seen below in Figure 3 is the chromatograms from sequencing. The results shows that the mutation GCC --> TTA was successful. The length of the sequence also suggest that amyloid-beta 1-42 was removed. Both parts was sequenced using the VR primer, and ~170 bases shorter fits completely with the length of the removed fragment. The full sequencing files including chromatograms can be found at BBa_K2671000.

Figure 3. The chromatograms from sequencing. Left is BBa_K2671000 (mutated part) and right is BBa_K2474000 (template).

References

[1] Part:BBa K2474000 - parts.igem.org [Internet]. [cited 2018 Oct 18]. Available from: http://parts.igem.org/Part:BBa_K2474000