Difference between revisions of "Part:BBa K3190601"

Revision as of 21:34, 21 October 2019

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

The coding sequence of BAX Inhibitor-I (BI-1) anti-toxin under the control of inducible promoter GAL1 (Basic part BBa_K3190050) 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.

Usage and Biology

Improvement of kill switch system

We seek 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 TEF1 constitutive promoter, the BI-1 coding sequence, and the yeast CYC1 terminator.

The kill switch proposed by team NAU China works by co-expressing the BAX gene (BBa_K2365048), 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.

Figure 1: Overview of the kill switch

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.

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.

BAX rescue assay

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).

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 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.

Sequence and Features