Template:NAU-CHINA/kill
Kill Switch
Bax, a member of the Bcl-2 family, is a mammalian derived pro-apoptotic protein that regulates apoptosis by promoting cell death. Bax can undergo different conformational changes, and its site of action appears to reside in mitochondria. BI-1 is an evolutionarily conserved integral membrane protein containing multiple membrane-spanning segments and is predominantly localized to intra-cellular membranes, similar to Bcl-2 family proteins. When over-expressed in yeast cells, BI-1 suppressed apoptosis included by Bax protein. In the kill switch design we will utilize the toxin-antitoxin (TA) system to functionally connect the two agonistic protein together to achieve our biosafety.
Approach
We utilized previous Bax protein plasmid and ameliorated it with a novel plant derived transcription factors (JUB1) that allows for orthologous and robust regulation of yeast viability. We chose to place one galactose regulated promoter (Gal1) upstream of the gene for the JUB1, which then act on the promoter of Bl-1. Consequently, the amount of transcription of Bl-1 is dependent on the concen-tration of JUB1. There is a constitutive promoter upstream the gene for the Bax protein. The ratio of Bl-1 and Bax is therefore engaging in the function of regulating yeast viability.
If there is no galactose in the circumstance, more Bax should be transcribed than Bl-1, so that Bax can serve the function of proapoptosis. When the yeasts’ life span are expected to be prolonged, we can activated the transcription of JUB1 by adding galactose in the circumstance. With the expression of JUB1, equal amounts of Bl-1 and Bax should be transcribed in yeasts cells and, therefore, Bl-1 and Bax should deactivate one another. As a result, Bax is unable to promote the proapoptosis of yeasts (i.e. yeasts can still undertake the function described in other modules).
Key Achievement
◎Successful validation of our modified protein—Bax mutant form1 possess a greater pro-apoptotic properties for yeast cells.
◎Successful validation of the Bax / Bl-1 suicide switch gene circuit can be induced by Saccharomyces cerevisiae.
◎Successful validation of JUB1 transcription can significantly deactivate the pro-apoptotic function of Bax protein.
Results
To test the effects of Bax, we insert the coding sequences of Bax into vectors pYES2 (URA as marker), subsequent to Gal1 promoter, so that Bax will be induced by galactose. We transformed the recombined plasmids pYES2-Bax into yeasts at the same time the pYES2 plasmid as a negative control.The yeasts containing pYES2-Bax or pYES2 were cultured to log phase in liquid URA-deficient SD media with 2% glucose as carbon sources, and were then dropped into liquid SD-URA-glucose(2%) media respectively and cultivated for one day. 10μl galactose aliquot was dropped on SD-URA-glucose(2%) plate periodically. The results are showed in Figure.1: As time passed, the amount of alive yeasts with pYES2-Bax was evidently decreased after the induction by galactose, and almost disappeared after 24 hours. In contrast, the yeasts with pYES2 as a negative control hardly reduced. This modified protein—Bax mutant form1 is our improvement on the previous part (BBa_K1061006) which is the coding sequence of hbax. To see more figures about our improvement please check out HERE!
Figure.1: 10μl galactose aliquot was treated with SD-URA-glucose(2%) plate periodically. The yeasts colonies were observed in a series of time gradients. It is evident that after the induction by galactose, and almost disappeared after 24 hours, while the negative control colony still stayed alive.
To prove the function of BI-1 protein, we construct an over-expression vector of BI-1, in which the transcription of BI-1 is started by a inducible promoter－JUB1p. We used the plasmid pRS423, which possess high copy number. Recombined plasmid pRS423-JUB1p-BI-1 was transformed into yeasts containing pYES2-Bax, and pRS423-JUB1p was also transformed. After the induction by galactose, OD450 of yeasts containing pRS423-JUB1p-BI-1/pYES2-Bax, pRS423-JUB1p/pYES2-Bax, and pYES2(used as control) were measured after CCK-8 staining respectively. The results are shown in Figure 2. Compared with modified Bax treated yeasts that possess the plasmid of pRS423-JUB1p-BI-1 can significantly prolong its life span.
Figure 2. After the induction by galactose, OD450 of yeasts containing pRS423-JUB1p-BI-1/pYES2-Bax, pRS423-JUB1p/pYES2-Bax, and pYES2 without any plasmids was used as control, Significant differences versus vehicle were assessed by student’s t test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=<0.0001.
