Difference between revisions of "Part:BBa K2365048:Experience"
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<b> pGAL1-BAX: Genomic integration vs. plasmid expression </b>  
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<b> <font size="4">Characterization of BAX: Genomic integration vs. plasmid expression</font> </b>
  
 
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.  
 
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.  
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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.  
 
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.  
  
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<b> Yeast transformation </b>  
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<b> <font size="4">Yeast transformation</font> </b>
  
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We confirmed the sequences of the pGAL1-BAX constructs cloned in <s>E. coli</s>, prior to transforming into <i>S. cerevisiae</i>.  
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After cloning and purifying the pGAL1-BAX constructs in <i>E. coli</i>, we confirmed the sequences of these, prior to transforming into <i>S. cerevisiae</i>.  
  
 
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.  
 
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.  
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<b> [INSERT GEL IMAGE of yeast colony PCR] </b>  
 
<b> [INSERT GEL IMAGE of yeast colony PCR] </b>  
  
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<small> <b> Figure 1: Yeast colony PCR </b> The positive colony of yeast is confirmed by the expected band size of around 1000 bp.</small>  
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<small> <b> Figure 1: Yeast colony PCR </b> | The positive colony of yeast is confirmed by the expected band size of around 1000 bp.</small>  
  
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For the plasmid expressing strain, we transformed the yeast with the GAL1-BAX cloned in a high copy plasmid (200 copies/cell) with a URA marker, as well
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For the plasmid expressing strain, we transformed the yeast with two plasmid: the GAL1-BAX cloned in a high copy plasmid (200 copies/cell) with a URA marker, and an empty vector plasmid with a TRP marker. After transforming the yeast, we grew the colonies on plates without URA and TRP, thus selecting for positive transformants. To further confirm the functionality of the GAL1 promoter, we also plated the transformed yeast on a plate with 1 % galactose. As expected, we did not see any growth on the galactose positive plate.
  
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picked the positive <i>E. coli</i> 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 <i>S. cerevisiae</i>. 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:
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[[File:PGAL1-BAX A.jpeg|300px]] [[File:PGAL1-BAX B.jpeg|300px]]
  
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<b> [Insert plate image of yeast 10] </b>
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<small><b>Figure 2: Transformant plates of dual plasmid transformed <i>S. cerevisiae</i></b> | Both transformed with pGAL1-BAX (URA marker) and an empty vector (TRP marker). A: plate with no galactose. B: plate with 1 % galactose. </small>
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<small>Figure legend: Transformant plates of dual plasmid transformed <i>S. cerevisiae</i>, transformed with pGAL1-BAX and an empty vector. Right: plate with 1 % galactose. Left: plate with no galactose. </small>
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<b> <font size="4">Galactose induction assay</font> </b>
  
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<b> Galactose induction assay </b>
 
 
To compare
 
To compare
  

Revision as of 17:22, 16 October 2019


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Applications of BBa_K2365048


Submission by Team UCopenhagen 2019:

Characterization of 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

After cloning and purifying the pGAL1-BAX constructs in E. coli, we confirmed the sequences of these, 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 1: Yeast colony PCR | The positive colony of yeast is confirmed by the expected band size of around 1000 bp.

For the plasmid expressing strain, we transformed the yeast with two plasmid: the GAL1-BAX cloned in a high copy plasmid (200 copies/cell) with a URA marker, and an empty vector plasmid with a TRP marker. After transforming the yeast, we grew the colonies on plates without URA and TRP, thus selecting for positive transformants. To further confirm the functionality of the GAL1 promoter, we also plated the transformed yeast on a plate with 1 % galactose. As expected, we did not see any growth on the galactose positive plate.

PGAL1-BAX A.jpeg PGAL1-BAX B.jpeg

Figure 2: Transformant plates of dual plasmid transformed S. cerevisiae | Both transformed with pGAL1-BAX (URA marker) and an empty vector (TRP marker). A: plate with no galactose. B: plate with 1 % galactose.


Galactose induction assay

To compare

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