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

 
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With the use of site-direted mutagenesis we wanted to improve the part of last years LiU iGEM team (BBa_K2474000). This was done by removing the fusion protein and the unnecessary bases as shown in figure 1. By doing this the fluorescent protein mNeonGreen (mNG) can be used as a reporter in a fully functional operone, with the use of the AraC/pBAD inducible system. The two additions was 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, we used SpeI 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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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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As seen in figure 2, our mutated version had a higher intensity on a UV-table.
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The results for our improved part can be seen in below. The reason we choose to improve this part was not only to create a functional reporter system, but also to illustrate the loss of exchange with a aggregation prone fusion tag. From fig 2. you can clearly see that the version without amyloid-beta 1-42 folds better and thus gives a much higher fluorescence, while the fusion version is having trouble.  
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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 was grown in LB-medium and 0.25 mg/ml L-arabinose 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 fig 2.
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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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<i>Figure 2. From left to right: Reference, BBa_K2671000 (mutated part) and BBa_K2474000.</i>
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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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Seen below in fig 3. is the chromatograms from sequencing. The results shows that the mutation GCC --> TAA 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 perfectly with the legnth of the removed fragment.  
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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>
 
<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