Template:NAU-CHINA/improved
IMPROVEMENT
Improve the function of an existing BioBrick Part ——BBa_K364202.
http://parts.igem.org/Part:BBa_K364202:Experience
Introduction
Coding sequence of Bax mutant form1 protein
Figure 1 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 SEY6 210 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.
Figure 7：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
Yeast ADH1 promoter
It is a strong constitutive yeast expression promoter
http://parts.igem.org/Part:BBa_J63005:Experience
Improved design
We construct the device：
ADH1 promoter+GFP+CYC1 terminator
ADH1 promoter is a truncated promoter, GFP is a mutant. These is a
invariable sequence upstream of GFP coding sequence.
We transformed this plasmid into Saccharomyces cerevisiae SEY6210.
Characterization
Figure 9: Fluorescence intensity of different colonies with truncated ADH1 promoter
The promoter of yeast alcohol dehydrogenase (ADH1) is widely used for the expression of heterologous genes in Saccharomyces cerevisiae. We tested the expression of GFP proteins in five transformants containing the ADH1 promoter. Our data shows that there are some differences in the fluorescence intensity of different yeast transformants with ADH1 promoter. The variation trends of promoter strengths from early-log to stationary phase varied in different transformants. Individual transformants colonies have different expression efficiency. We will test this promoter for more than three times.
TDH3 promoter
It is a strong constitutive yeast expression promoter
http://parts.igem.org/Part:BBa_K530008:Experience
Improved design
We construct the device：
TDH3 promoter+GFP+ADH1 terminator
TDH3 promoter is a truncated promoter, GFP is a mutant. These is a invariable sequence upstream of GFP coding sequence.
Characterization
Figure 10. Fluorescence intensity of different colonies of TDH3 promoter and improved TDH3 promoter.
The responses of promoter strengths of artificial TDH3 promoter and native promoter TDH3 were comprehensively compared. The strength of improved TDH3 was always higher than native promoter TDH3，but varied along with the genetic background of host.
