Part:BBa_K1061006

Bax mutant form

       Hbax, a member of Bcl-2 family, is an apoptosis-related protein. It integrates into the 

mitochondrial outer membrane, releases cytochrome C and leads to apoptosis.Hbax184a is a mutant of hbax that constantly integrates into mitochondrial outer membrane. We have tried to use them as suicide genes, but they can not induce apoptosis in most cancer cell lines, probably because the pathway that they involve has been blocked in most cancer cells. However, we find that the protein can induce “apoptosis” in the most broadly used eukaryotic chassis, yeast. So it will be very potential to use the gene in some circuit that need to kill the yeast in proper condition. 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 452
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Functional experiment

    Hbax mutant under indution of galactose
    This is the pre-experment that we done.We inserted the hbax mutant form under the control of 
Gal promoter, then put it under the induction of the galatose. From left to right, the induction 

concentration is 0, 1%, and 2%. We can see a significant decrease of yeast concentration when put it in a high level of induction(2%)

pre-gradient experiment that we have done.

quantutative analysis

  We have count the cell density of the yeast in the tude under 3 concentration, for the picture

below,top to down:0,1%,2%, it should be some treashold for the indution of apoptosis, so the gene behave somehow like a switch.

pre-gradient experiment that we have done data.

the data reveal in a picture form.

And, this year , SCUT also help us to characterize the part, by comparing its growth-inhibiting effect with the original hbax that we improve, we can see from the picture that the inhibition level of hbax mutant(label as hbaxs)is significantly higher than the hbax already exist.And they can both inhibit the growth comparing to the control. However, since 2% galactose is a very high indution level, it turns out that the highest expression level already reach at this point, so there are almost no difference between 2% and 5%. For more acurate gradient data, we need to set the gradient between 1% and 2%

the growth curve.

Kill range

For each suicide gene, it is important to know which cancer cells and normal cells it will kill.So that people can speficially choose gene that will be functional in their project when duelling about cancer. And we define this new feature for each suicide gene that we submitted. The list will be changed due to more and more experiment will be done. We highly recommend people to adopt this feature as an important characteristic of suicide gene.