Difference between revisions of "Part:BBa K2365048:Experience"

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Applications of BBa_K2365048

Submission by Team UCopenhagen 2019:

pGAL1-BAX: Genomic integration vs. plasmid expression

We further characterized the Bax protein under the expression of an inducible promoter, pGAL1 (BBa_K3190050). We have characterized the apoptotic potential of Bax protein, when the BAX gene is integrated into the yeast genome, as compared to when it is expressed on a high copy plasmid.

Using USER ligation, we assembled the BAX gene with pGAL1 on a plasmid backbone compatible with multiplex integration cassette. The backbone used contains a URA selection marker, and will integrate the construct in the yeast genome at chromosome 11, site 2.

Furthermore, we also transformed yeast with a dual plasmid system. Using USER ligation, we assembled pGAL1 and BAX on a high copy plasmid backbone (200 copies/cell) containing a URA selection marker.

Yeast transformation

We confirmed the sequences of the pGAL1-BAX constructs, both genomically integrated, and plasmid expressed prior to transforming into S. cerevisiae.

For the genomically integrated strain, the positive transformants were confirmed by performing yeast colony PCR. We used 2 primers, one in the forward direction for the backbone and one in the reverse direction for the yeast chromosome 11. In the presence of our construct, we expect to see a band at around 1000 bp as, that is the size of the fragment between the two primer regions. In the absence of the constructs, we expect to see the bands at around 1500 bp, as this is the size of site 2 of chromosome 11.

[INSERT GEL IMAGE of yeast colony PCR] Figure Legend: Above gel image shows the positive colony of yeast successfully transformed with the pGAL1-BAX construct. We see the expected band size of around 1000 bp.

Yeast transformation: dual plasmid transformation We picked the positive E. coli colonies of the pGAL1-BAX cloned in a high copy plasmid, and purified the DNA from these. After confirming the sequence, we transformed the construct into S. cerevisiae. To select for positive transformants, we also transformed an empty vector with a TRP selection marker, and grew the colonies on plates without both URA and TRP. As a control, we spread the cells on plates both with and without galactose. No growth was detected in the galactose containing plate, as seen below:

[Insert plate image of yeast 10] Figure legend: Transformant plates of dual plasmid transformed S. cerevisiae, transformed with pGAL1-BAX and an empty vector. Right: plate with 1 % galactose. Left: plate with no galactose.

Galactose induction assay To compare

User Reviews

UNIQa15fba099b5bf543-partinfo-00000001-QINU UNIQa15fba099b5bf543-partinfo-00000002-QINU

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 ===Applications of BBa_K2365048===
-Coding sequence of the mammalian apoptosis regulator Bax protein. The gene will be integrated into the genome of the fungal chassis <i>Saccharomyces cerevisiae</i>, 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 the part orginally submitted by iGEM17_NAU-CHINA.
+<br>
+<b> <font size="5">Submission by Team UCopenhagen 2019:</font> </b>
+<hr>
-===Usage and Biology===
+<b> pGAL1-BAX: Genomic integration vs. plasmid expression </b>
-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.
+We further characterized the Bax protein under the expression of an inducible promoter, pGAL1 (<partinfo>BBa_K3190050</partinfo>). We have characterized the apoptotic potential of Bax protein, when the BAX gene is integrated into the yeast genome, as compared to when it is expressed on a high copy plasmid.
-[[File:UCopenhagen Killswitch.jpeg|600px]]
-<small> Figure legend: Overview of the 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 contains a URA selection marker, and will integrate the construct in the yeast genome at chromosome 11, site 2.
-<b> <i>E. coli</i> cloning of pGAL1-BAX </b>
+Furthermore, we also transformed yeast with a dual plasmid system. Using USER ligation, we assembled pGAL1 and BAX on a high copy plasmid backbone (200 copies/cell) containing a URA selection marker.
-The construct was successfully cloned in E. coli as confirmed by below gel image of colony PCR. We used forward primer for the promoter (pGAL1) and reverse primer for the gene (BAX). We therefore expect a band of 1034 bp.
-<b>[INSERT GEL IMAGE!!] </b>
-<small>Figure Legend: Above gel electrophoresis image shows the positive colony PCR sample. A band was observed around 1000 bp, which correlates well to the expected band size for a construct of 1034 bp. </small>
-In order to further characterize the expression of BAX under pGAL1, we also transformed yeast with a dual plasmid system. Using USER ligation, we assembled pGAL1 and BAX on a high copy plasmid backbone containing a URA selection marker. The construct was successfully cloned in <i>E. coli</i>, as depicted by below colony PCR gel image:
-[Insert pUUS gel image]
-<small>Figure legend: Gel image showing the positive <i>E. coli</i> colonies of pGAL1-BAX. The expected band is seen around 1000 bp.</small>
-<b> Yeast transformation: genome integration </b>
+<b> Yeast transformation </b>
-For the yeast transformation, we picked the positive <i>E. coli </i> colonies and purified DNA from these. After confirming the sequence, we successfully transformed the construct into <i>S. cerevisiae</i> as depicted in below gel image from yeast colony PCR.
+We confirmed the sequences of the pGAL1-BAX constructs, both genomically integrated, and plasmid expressed prior to transforming into <i>S. cerevisiae</i>.
-For the colony PCR, we used 2 primers, one in the forward direction for the backbone and one in the reverse direction for the yeast chromosome 11. In the presence of our construct, we expect to see a band at around 1000 bp as, that is the size of the fragment between the two primer regions. In the absence of the constructs, we expect to see the bands at around 1500 bp, as this is the size of site 2 of chromosome 11.
+For the genomically integrated strain, the positive transformants were confirmed by performing yeast colony PCR. We used 2 primers, one in the forward direction for the backbone and one in the reverse direction for the yeast chromosome 11. In the presence of our construct, we expect to see a band at around 1000 bp as, that is the size of the fragment between the two primer regions. In the absence of the constructs, we expect to see the bands at around 1500 bp, as this is the size of site 2 of chromosome 11.
 <b> [INSERT GEL IMAGE of yeast colony PCR] </b>