Difference between revisions of "Part:BBa K3190601"
 
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We wanted to improve the BI-1 coding sequence (<partinfo>BBa_K2365518</partinfo>), which was submitted by the iGEM team NAU_CHINA 2017. They used it as part of their kill switch system and also submitted a composite part consisting of the TEF1 constitutive promoter, BI-1, and the yeast CYC1 terminator.  
 
We wanted to improve the BI-1 coding sequence (<partinfo>BBa_K2365518</partinfo>), which was submitted by the iGEM team NAU_CHINA 2017. They used it as part of their kill switch system and also submitted a composite part consisting of the TEF1 constitutive promoter, BI-1, and the yeast CYC1 terminator.  
  
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The kill switch proposed by team NAU China works by co-expressing the BAX gene (<partinfo>BBa_K2365048</partinfo>), which encodes the pro-apoptotic Bax protein, under a constitutive promoter, together with BI-1 under the galactose inducible GAL1 promoter. In theory, as long as galactose is present in the media BI-1 will be expressed, inhibiting the Bax protein. Should the yeast cell escape the media or matrix, BAX will be expressed, causing apoptosis of the cell.  
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The kill switch proposed by team NAU China would work by co-expressing the BAX gene (<partinfo>BBa_K2365048</partinfo>), which encodes the pro-apoptotic BAX protein, under a constitutive promoter, together with BI-1 under the galactose inducible GAL1 promoter. In theory, as long as galactose is present in the media, BI-1 will be expressed, inhibiting the BAX protein. Should the yeast cell escape the media or matrix, BAX will be expressed, causing apoptosis of the cell.  
  
 
[[File:UCopenhagen Killswitch.jpeg|600px]]
 
[[File:UCopenhagen Killswitch.jpeg|600px]]
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<small><b>Figure 1: Overview of the kill switch </b></small>
 
<small><b>Figure 1: Overview of the kill switch </b></small>
  
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Our improvement lays in the addition of the pGAL1 promoter in front of the BI-1 sequence. This improvement will allow us to control the expression of BI-1, as the GAL1 promoter allows transcription of BI-1 only in the presence of galactose.  
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We improved the existing BI-1 sequence by expressing it under the control of the inducible pGAL1 promoter. This improvement will allow us to control the expression of BI-1, as the GAL1 promoter allows transcription of BI-1 only in the presence of galactose.  
  
  
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In order to show that BI-1 is able to neutralize the lethal impact of BAX, we performed a rescue assay in which both BAX and BI-1 were placed under the control of the GAL1 promoter. For this, we constructed two strains; OV19, which had pGAL1-BAX integrated into the genome and pGAL1-BI-1 on a high-copy number episomal plasmid, and OV20, which had pGAL1-BAX integrated into the genome and carried an empty vector.
 
In order to show that BI-1 is able to neutralize the lethal impact of BAX, we performed a rescue assay in which both BAX and BI-1 were placed under the control of the GAL1 promoter. For this, we constructed two strains; OV19, which had pGAL1-BAX integrated into the genome and pGAL1-BI-1 on a high-copy number episomal plasmid, and OV20, which had pGAL1-BAX integrated into the genome and carried an empty vector.
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For the assay colonies were picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalized to 0.035 and different dilutions of the colonies (10^-0 to 10^-3) were spotted on plates with glu-U-W agar and raff-U-W agar with 1% galactose in volumes of 10 µl. A yeast strain containing only pGAL1-BAX and the empty pWUS plasmid served as control (OV20) and was spotted along with the sample (Figure 2).  
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For the assay colonies were picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalized to 0.035 and different dilutions of the colonies (10<sup>-0</sup> to 10<sup>-3</sup>) were spotted on plates with glu-U-W agar and raff-U-W agar with 1% galactose in volumes of 10 µl. A yeast strain containing only pGAL1-BAX and the empty pWUS plasmid served as control (OV20) and was spotted along with the sample (Figure 2).  
  
  
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[[File:ovulaid35.png|500px]]
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[[File:ovulaid35.png|800px]]
  
 
<small><b>Figure 2: BAX rescue assay</b> | In this assay, the strains OV19 and OV20 were grown on both glu-U-W agar (left) and raff-U-W (1% galactose) agar in different dilutions of OD600nm (10-0 to 10-3). </small>
 
<small><b>Figure 2: BAX rescue assay</b> | In this assay, the strains OV19 and OV20 were grown on both glu-U-W agar (left) and raff-U-W (1% galactose) agar in different dilutions of OD600nm (10-0 to 10-3). </small>

Latest revision as of 00:27, 22 October 2019


pGAL1-BI-I: BI-I CDS under inducible promoter

Coding sequence of BAX Inhibitor-I (BI-1) anti-toxin under control of the galactose inducible GAL1 promoter (BBa_K3190050). This biobrick was designed for use in a yeast kill switch system, as described by NAU_CHINA 2017 (Figure 1).

Usage and Biology

Improvement of kill switch system

We wanted to improve the BI-1 coding sequence (BBa_K2365518), which was submitted by the iGEM team NAU_CHINA 2017. They used it as part of their kill switch system and also submitted a composite part consisting of the TEF1 constitutive promoter, BI-1, and the yeast CYC1 terminator.

The kill switch proposed by team NAU China would work by co-expressing the BAX gene (BBa_K2365048), which encodes the pro-apoptotic BAX protein, under a constitutive promoter, together with BI-1 under the galactose inducible GAL1 promoter. In theory, as long as galactose is present in the media, BI-1 will be expressed, inhibiting the BAX protein. Should the yeast cell escape the media or matrix, BAX will be expressed, causing apoptosis of the cell.

UCopenhagen Killswitch.jpeg

Figure 1: Overview of the kill switch

We improved the existing BI-1 sequence by expressing it under the control of the inducible pGAL1 promoter. This improvement will allow us to control the expression of BI-1, as the GAL1 promoter allows transcription of BI-1 only in the presence of galactose.


BAX rescue assay

In order to show that BI-1 is able to neutralize the lethal impact of BAX, we performed a rescue assay in which both BAX and BI-1 were placed under the control of the GAL1 promoter. For this, we constructed two strains; OV19, which had pGAL1-BAX integrated into the genome and pGAL1-BI-1 on a high-copy number episomal plasmid, and OV20, which had pGAL1-BAX integrated into the genome and carried an empty vector. For the assay colonies were picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalized to 0.035 and different dilutions of the colonies (10-0 to 10-3) were spotted on plates with glu-U-W agar and raff-U-W agar with 1% galactose in volumes of 10 µl. A yeast strain containing only pGAL1-BAX and the empty pWUS plasmid served as control (OV20) and was spotted along with the sample (Figure 2).


Ovulaid35.png

Figure 2: BAX rescue assay | In this assay, the strains OV19 and OV20 were grown on both glu-U-W agar (left) and raff-U-W (1% galactose) agar in different dilutions of OD600nm (10-0 to 10-3).

As BAX was expressed under the inducible promoter pGAL1 in both strains, we expected normal growth on the glu-U-W plates. On the raff-U-W plates with 1% galactose, we expected the control strain OV20 to show decreased colony size and number compared to the OV19 strain that should be rescued by the BI-1 plasmid. However, as seen on figure 2, no significant growth was seen for either strain in the presence of galactose. As such, we saw no indication that BI-1 was able to prevent BAX induced apoptosis. While it is possible that the lack of function of BI-1 could be the result of a loss of function during transformation, our data suggests BI-1 is not able to prevent BAX induced apoptosis in our system, and is therefore not suitable for our kill switch.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 954
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 377
  • 1000
    COMPATIBLE WITH RFC[1000]