Difference between revisions of "Part:BBa K1061006"
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| − | + | __NOTOC__ | |
<partinfo>BBa_K1061006 short</partinfo> | <partinfo>BBa_K1061006 short</partinfo> | ||
| − | 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. | + | 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 | ||
| + | |||
| + | <!-- Add more about the biology of this part here | ||
| + | ===Usage and Biology=== | ||
| + | <!-- --> | ||
| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K1061006 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | ====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 | 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 | 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 | 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%) | it in a high level of induction(2%) | ||
| − | [[Image:Hbax pre-experiment.png|thumb|center| | + | [[Image:Hbax pre-experiment.png|thumb|center|800px|pre-gradient experiment that we have done.]] |
| + | |||
====quantutative analysis==== | ====quantutative analysis==== | ||
We have count the cell density of the yeast in the tude under 3 concentration, for the picture | 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 | 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. | behave somehow like a switch. | ||
| − | [[Image:Hbax_yeast_cell_count.png|thumb|center| | + | [[Image:Hbax_yeast_cell_count.png|thumb|center|800px|pre-gradient experiment that we have done data.]] |
| + | |||
| + | [[Image:Hbax_data.jpg|thumb|center|800px|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% | ||
| + | [[Image:Hbax_and_hbax_mutant_comparing.jpg|thumb|center|800px|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. | ||
| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K1061003 parameters</partinfo> | ||
| + | <!-- --> | ||
| + | <h2>References:[1]Qunli Xu et al,Bax Inhibitor-1, a Mammalian Apoptosis Suppressor Identified by Functional Screening in Yeast,molecular cell,1,3,337-346,1998</h2> | ||
| + | <p> | ||
Latest revision as of 11:37, 2 October 2013
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
- 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]
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%)
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.
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%
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.