Difference between revisions of "Part:BBa K3190601"

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.

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).

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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3190601 short</partinfo>
-The coding sequence of BAX Inhibitor-I (BI-1) anti-toxin under the control of inducible promoter GAL1 (Basic part <partinfo>BBa_K3190050</partinfo>) for the design of a kill switch system for a yeast wherein a BAX toxin is expressed under a constitutive promoter. This system ensures that the yeast will not survive if it escapes the medium containing the inducer.
+Coding sequence of BAX Inhibitor-I (BI-1) anti-toxin under control of the galactose inducible GAL1 promoter (<partinfo>BBa_K3190050</partinfo>). This biobrick was designed for use in a yeast kill switch system, as described by NAU_CHINA 2017 (Figure 1).
 ===Usage and Biology===
 <b><font size="3">Improvement of kill switch system</font></b>
-We seek 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 TEF1 constitutive promoter, the BI-1 coding sequence, 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.
-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, and BI-1 under the inducible GAL1 promoter. 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.
+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]]
-<small><b>Figure 1:</b> Overview of the kill switch. </small>
+<small><b>Figure 1: Overview of the kill switch </b></small>
-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.
+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.
-this improvement is necessary to use this system as a kill switch and that the composite Biobrick that NAU-china 2017 themselves submitted is not able to allow apoptosis, since  they had a TEF1 promoter controlling BI-1 which is not dependent the environmental galactose amount.
+<b><font size="3">BAX rescue assay</font></b>
-<b><font size="3">BAX rescue assay</font></b>
+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<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).
-To show that BI-1 is able to neutralise the lethal impact of BAX on our yeast, we have performed a rescue assay by means of putting both BAX and BI-1 under the GAL1 promoter (OV19).
-For construction of the strain, we have used the yeast strain with the genome integrated pGAL1-BAX (OV6) and transformed it with pGAL1-BI-1 in the previously used pWUS plasmid.
-Transformants were then picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalised 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).
-[[File:ovulaid35.png|500px]]
+[[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>
-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 the strain OV19 that should be rescued by the BI-1 plasmid. However, figure 24 shows that there is no significant growth for either of the strains in the presence of galactose meaning that as it stands now, we can conclude that the colonies that have been picked for this assay were either not transformed with BI-1 (and only kept the markers) or BI-1 is not able to prevent BAX induced apoptosis in our yeast strain and is therefore not suitable for our kill switch.
+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.