Difference between revisions of "Part:BBa K3190501"

Revision as of 08:44, 15 October 2019

BAX: Bcl-2-associated X protein CDS

Coding sequence of the mammalian apoptosis regulator Bax protein. The gene will be integrated into the genome of the fungal chassis Saccharomyces cerevisiae, acting as the key gene in our biosafety device. Overexpression of Bax is lethal in S. cerevisiae, leading to the release of cytochrome c from mitochondria and thus apoptosis. The following results are characterization of a part submitted by iGEM17_NAU-CHINA: BBa_K2365048.

Usage and Biology

For our biosafety device, the Bax protein will be under the expression of a constitutive promoter, pADH2 (No part name specified with partinfo tag.). An anti-toxin, BAX Inhibitor-1 (BI-1) (BBa_K3190502) will be under the expression of an inducible promoter, pGAL1 (BBa_K3190050). As long as there is galactose present in the media, the anti-toxin will be expressed, inhibiting the apoptotic effects of Bax protein. If the cell escapes the media, the anti-toxin will no longer be expressed, and Bax protein will cause apoptosis of the cell.

Figure legend: Overview of our toxin/anti-toxin biosafety device.

Designing the construct

In order to characterize the Bax protein, we cloned it to inducible promoter pGAL1 (BBa_K3190050), as expression under a constitutive promoter would be expected to cause the cells to die.

Using USER ligation, we assembled the BAX gene with pGAL1 on a plasmid backbone compatible with multiplex integration cassette. The backbone used will integrate the construct in the yeast genome at chromosome 11, site ???.

E. coli cloning of pGAL1-BAX

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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3190501 short</partinfo>
-Coding sequence of the mammalian apoptosis regulator Bax protein. The gene will be integrated into the genome of the fungal chassis Saccharomyces cerevisiae, acting as the key gene in our biosafety device. Overexpression of Bax is lethal in S. cerevisiae, leading to the release of cytochrome c from mitochondria and thus apoptosis.
+Coding sequence of the mammalian apoptosis regulator Bax protein. The gene will be integrated into the genome of the fungal chassis Saccharomyces cerevisiae, acting as the key gene in our biosafety device. Overexpression of Bax is lethal in <i>S. cerevisiae</i>, leading to the release of cytochrome c from mitochondria and thus apoptosis. The following results are characterization of a part submitted by iGEM17_NAU-CHINA: <partinfo>BBa_K2365048</partinfo>.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+For our biosafety device, the Bax protein will be under the expression of a constitutive promoter, pADH2 (<partinfo>BBa_K3190006</partinfo>). An anti-toxin, BAX Inhibitor-1 (BI-1) (<partinfo>BBa_K3190502</partinfo>) will be under the expression of an inducible promoter, pGAL1 (<partinfo>BBa_K3190050</partinfo>). As long as there is galactose present in the media, the anti-toxin will be expressed, inhibiting the apoptotic effects of Bax protein. If the cell escapes the media, the anti-toxin will no longer be expressed, and Bax protein will cause apoptosis of the cell.
+[[File:UCopenhagen Killswitch.jpeg|600px]]
+<small> Figure legend: Overview of our toxin/anti-toxin biosafety device. </small>
+<b> Designing the construct </b>
+In order to characterize the Bax protein, we cloned it to inducible promoter pGAL1 (<partinfo>BBa_K3190050</partinfo>), as expression under a constitutive promoter would be expected to cause the cells to die.
+Using USER ligation, we assembled the BAX gene with pGAL1 on a plasmid backbone compatible with multiplex integration cassette. The backbone used will integrate the construct in the yeast genome at chromosome 11, site ???.
+<b> <i>E. coli</i> cloning of pGAL1-BAX </b>
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