Part:BBa_K2365048
Bax mutant form1
Coding sequence of Bax mutant form1 protein which releases cytochrome C and leads to apoptosis in the most broadly used eukaryotic chassis. This year we choose Saccharomyces cerevisiae as our chassis fungal, so we use this gene as the key gene in our biosafety device. It worked well when we detected it's function.We submitted as a improvement of already exist hbax biobrick. The link of original biobrick is here:https://parts.igem.org/Part:BBa_K364202
Characterization & Improvement by AFCM-Egypt 2021
Characterization Of Mutational Landscape
Team:AFCM-Egypt 2021has characterized & improved this part As shown in BBa_K3743013 by performing mutagenesis prediction of mutational landscape of Bax mutant and testing the effect of these mutations on the evolutionary fitness of the protein after generating multiple sequence alignment of the protein sequence and predict mutational landscapes. As shown in the chart, the (G36E) mutation showed the highest score compared to other mutations. On the contrary, we can see that the (G36K) contributed to the lowest evolutionary fitness to Bax mutant. As shown in Figure (1), That's why we changed the sequence according to the highest positive mutation.
Improvement of RFC [21] Assembly compatibility
We improved BAX sequence by performing codon optimization on parts of the sequence that would be a target of restriction enzyme BamHI making it a better fit for assembly as shown in part BBa_K3743013
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 452
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction：
Coding sequence of Bax mutant form1 protein.
Figure1: Picture of gel electrophoresis (PCR validated products of Bax mutant form1 ; 579bp). Function：We use Bax protein to kill our engineered yeast when the fungal has finished its mission.
Improved design:
The 579bp fragment was cloned under the GAL1 promoter, followed by CYC1 terminator. The pYES2 vector is designed for native expression of protein of interest in S. cerevisiae. It contains the URA3 gene for selection in yeast and 2µ origin for high-copy maintenance. After verifying the right constructs by restriction enzyme digestions and sequencing, the plasmids were transformed into S. cerevisiae SEY6210.
Characterization：
Figure 2: Yeast growth curve induced by galactose. The expression of Bax protein was activated by inducer-2% galactose. The cells were washed and diluted 1:1000 into fresh medium to induce the GAL promoter. We use the microplate reader to test the value of OD600. By comparing pYES2-Bax’s growth-inhibiting effect with the control, we can see from the picture that the inhibition level of Bax mutant form1 induced by 2% galactose is significantly higher than the control. It is remarkable that expression of the proapoptotic protein Bax conferred a lethal phenotype in the yeast.
Figure 3: Yeast growth curve (with glucose as carbon source). In theory, when there is glucose, Bax protein is not expressed. However, from the picture we can be seen that there is a certain leakage.
Apoptotic phenotype in Saccharomyces cerevisiae SEY6210 induced by Bax protein:
Figure 4: We can see a significant decrease of yeast concentration.After centrifugation,fewer precipitated cells of induced medium were seen than that of uninduced medium.
We have count the cell density of the yeast:
Figure 5 :Observing yeast cells under a microscope.
Figure 6 : Typan Blue Staining Cell Viability Assay . The stained cells in the view are dead cells.
CCK-8 testing
Method: Cells were added into 96-well microplates. Then CCK8 solution were added to the wells and the plates were incubated for additional 4 h at 30°C. Optical density was measured using a microplate reader at a wavelength of 450 nm.
Figure7:Comparison between two groups was performed using Student's t-test. Results are expressed as mean ± SD of 3 independent experiments. Differences between means were considered significant when the two-tailed P-value was <0.05.
Apoptotic features of Bax-expressing yeast:
Figure 8: Bax-expressing yeast, the number is getting less and less over time.
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