Difference between revisions of "Part:BBa K2365048"
(→300px|center) |
(→300px|center) |
||
| Line 25: | Line 25: | ||
===[[File:图片4.png|300px|center]]=== | ===[[File:图片4.png|300px|center]]=== | ||
| + | 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. | ||
| + | |||
===[[File:图片5.png|300px|center]]=== | ===[[File:图片5.png|300px|center]]=== | ||
===[[File:图片6.png|300px|center]]=== | ===[[File:图片6.png|300px|center]]=== | ||
Revision as of 10:54, 31 October 2017
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
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]
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.
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.
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.
