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===Applications of BBa_K2365048===
 
===Applications of BBa_K2365048===
  
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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.
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<br>
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<b> <font size="5">Submission by Team UCopenhagen 2019:</font> </b>
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<hr>
  
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===Usage and Biology===
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We further characterized the Bax protein under the expression of an inducible promoter, pGAL1 (<partinfo>BBa_K3190050</partinfo>). We chose to use this part, as it is Team NAU China 2017's improved version of the original part of Team Debrecen-Hungary 2010 (<partinfo>BBa_K364202</partinfo>).
  
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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.  
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As part of our studies we both integrated BAX into the chromosome of <i>Saccharomyces cerevisiae</i> and expressed it on an episomal
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plasmid. In both cases we found that increasing galactose concentrations resulted in reduction in growth.
  
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[[File:UCopenhagen Killswitch.jpeg|600px]]
 
  
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<small> Figure legend: Overview of the toxin/anti-toxin biosafety device. </small>
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<b> <font size="4">Yeast transformation</font> </b>
  
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<b> Designing the construct </b>
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Initially, our yeast was transformed with episomal plasmids containing BAX under a galactose inducible promoter. For our control strain we used an empty vector. Following transformation, each culture were split into two. One half was plated on a plate containing raffinose selection media with 0.0 % galactose, and the other half on raffinose selection media containing 1.0 % galactose. In both strains a second empty vector was introduced, placing both strains under Uracil (U) and Tryptophan (W) selection.
  
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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.  
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As can be seen on Figure 2, no colonies were visible on the plate containing galactose, suggesting that the BAX protein was expressed in such a high concentration that all cells died. For our control strain an equal amount of cells were seen both with and without galactose.
  
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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.
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For more stable expression, we also interated pGAL1-BAX into the chromosome of <i>S. cerevisiae</i>, using U selection. Here, positive transformants were selected for by performing yeast colony PCR (Figure 1). This strain was used for all further experiments.
  
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<b> <i>E. coli</i> cloning of pGAL1-BAX </b>
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[[File:ovulaid29.jpg|300px]]
  
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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.
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<small> <b> Figure 1: Yeast colony PCR of genomically integrated transformants</b> | The positive colony of yeast is confirmed by the expected band size of around 800 bp.</small>
  
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<b>[INSERT GEL IMAGE!!] </b>
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[[File:PGAL1-BAX A.jpeg|300px]] [[File:PGAL1-BAX B.jpeg|300px]]
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<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>
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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:
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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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[Insert pUUS gel image]
 
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<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>
 
  
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<b> Yeast transformation: genome integration </b>  
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<b> <font size="4">Galactose induction assay</font> </b>
  
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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.  
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To analyse the effect of BAX on yeast under different expression levels, we conducted a galactose induction assay using raff-U plates with five different galactose concentrations.
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Cultures of yeast containing pGAL1-BAX or empty vector were grown O/N and then diluted to an OD<sub>600nm</sub> of 0.5. On plates with either 0%, 0.025%, 0.05%, 0.1% or 0.2% galactose, 10 µl of each culture were spotted in increasing dilutions (10<sup>-1</sup> to 10<sup>-4</sup>; Figure 3 and 4) and incubated for three days at 30 °C. The strain with the integrated pGAL1-BAX construct showed decreased growth compared to the control strain even when galactose was absent (Figure 3). This suggests that the galactose promoter is leaky and a low amount of BAX is produced at all times.
  
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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.  
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[[File:ovulaid31.png|600px]]
  
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<b> [INSERT GEL IMAGE of yeast colony PCR] </b>
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<small><b>Figure 3: Growth of pGAL1-BAX and the empty vector strain in the absence of galactose. </b>The cultures were spotted in the dilutions 10<sup>-1</sup> to 10<sup>-4</sup> of an OD<sub>600nm</sub> of 0.5 and incubated for three days at 30 °C. </small>
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<small> 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.</small>  
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<b> Yeast transformation: dual plasmid transformation </b>
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Moreover, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed (Figure 4) when comparing the spots of the pGAL1-BAX strain at a dilution of 10<sup>-1</sup> at different galactose concentrations. This suggests that successful induction of BAX leads to apoptosis in our yeast.
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We 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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<b> [Insert plate image of yeast 10] </b>
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[[File:ovulaid32.png|600px]]
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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> Galactose induction assay </b>
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<small><b>Figure 4: Growth comparison of pGAL1-BAX and control strain in the presence of varying galactose concentrations.</b> | Shown are the CFUs at an OD<sub>600nm</sub> of 0.05 after incubation at 30 °C for four days. The three yellow colonies seen at 0.2% galactose on the pGAL-BAX plate can be morphologically distinguished from the others, suggesting that they are contaminants.</small>
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To compare
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<br>
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<b> <font size="4">Quantitative galactose induction assay</font> </b>
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To further analyze the effect of BAX on our yeast under different expression levels, we conducted a quantitative galactose induction assay. Here, an O/N culture of yeast containing pGAL1-BAX was diluted to an OD<sub>600nm</sub> of 0.5. Subsequently, 100 µl of the culture in dilutions of 10<sup>-3</sup> and 10<sup>-4</sup> were spread on plates with 0%, 0.05%, 0.1%, 0.3% and 1% galactose, respectively. Each plate was made in duplicate. After incubation for three days at 30 °C, the CFU/ml were calculated and compared to the control (Figure 5).
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<b> [Discussion of below graph] </b>
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[[File:ovulaid30.png|600px]]
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<small><b>Figure 5: Gradient induction of BAX in yeast using the inducible GAL1 promoter.</b> | If no errorbar is indicated, only one of the used duplets showed a quantifiable amount of colonies. </small>
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As seen on figure 6, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately our control strain was only plated on raff-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to pGAL1-BAX grown in 0 % galactose media).
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In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this that the cells were killed before a normal sized colony could be formed. To test this, the pGAL1-BAX plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed (Figure 6).
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[[File:ovulaid33.png|600px]]
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<small><b>Figure 6: Comparison of colony sizes of pGAL1-BAX and the control containing the empty vector grown on raff-U agar with galactose.</b> | Left: Colonies of the control strain  (dilution  10<sup>-3</sup>) in the presence of 1% galactose after incubation for three days at 30 °C. Right: Colonies of the pGAL1-BAX strain (dilution  10<sup>-3</sup>) in the presence of 0.1% galactose after incubation for four days at 30 °C. </small>
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 23:32, 21 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2365048


Submission by Team UCopenhagen 2019:

We further characterized the Bax protein under the expression of an inducible promoter, pGAL1 (BBa_K3190050). We chose to use this part, as it is Team NAU China 2017's improved version of the original part of Team Debrecen-Hungary 2010 (BBa_K364202).

As part of our studies we both integrated BAX into the chromosome of Saccharomyces cerevisiae and expressed it on an episomal plasmid. In both cases we found that increasing galactose concentrations resulted in reduction in growth.


Yeast transformation

Initially, our yeast was transformed with episomal plasmids containing BAX under a galactose inducible promoter. For our control strain we used an empty vector. Following transformation, each culture were split into two. One half was plated on a plate containing raffinose selection media with 0.0 % galactose, and the other half on raffinose selection media containing 1.0 % galactose. In both strains a second empty vector was introduced, placing both strains under Uracil (U) and Tryptophan (W) selection.

As can be seen on Figure 2, no colonies were visible on the plate containing galactose, suggesting that the BAX protein was expressed in such a high concentration that all cells died. For our control strain an equal amount of cells were seen both with and without galactose.

For more stable expression, we also interated pGAL1-BAX into the chromosome of S. cerevisiae, using U selection. Here, positive transformants were selected for by performing yeast colony PCR (Figure 1). This strain was used for all further experiments.

Ovulaid29.jpg

Figure 1: Yeast colony PCR of genomically integrated transformants | The positive colony of yeast is confirmed by the expected band size of around 800 bp.

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 analyse the effect of BAX on yeast under different expression levels, we conducted a galactose induction assay using raff-U plates with five different galactose concentrations. Cultures of yeast containing pGAL1-BAX or empty vector were grown O/N and then diluted to an OD600nm of 0.5. On plates with either 0%, 0.025%, 0.05%, 0.1% or 0.2% galactose, 10 µl of each culture were spotted in increasing dilutions (10-1 to 10-4; Figure 3 and 4) and incubated for three days at 30 °C. The strain with the integrated pGAL1-BAX construct showed decreased growth compared to the control strain even when galactose was absent (Figure 3). This suggests that the galactose promoter is leaky and a low amount of BAX is produced at all times.

Ovulaid31.png

Figure 3: Growth of pGAL1-BAX and the empty vector strain in the absence of galactose. The cultures were spotted in the dilutions 10-1 to 10-4 of an OD600nm of 0.5 and incubated for three days at 30 °C.

Moreover, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed (Figure 4) when comparing the spots of the pGAL1-BAX strain at a dilution of 10-1 at different galactose concentrations. This suggests that successful induction of BAX leads to apoptosis in our yeast.

Ovulaid32.png

Figure 4: Growth comparison of pGAL1-BAX and control strain in the presence of varying galactose concentrations. | Shown are the CFUs at an OD600nm of 0.05 after incubation at 30 °C for four days. The three yellow colonies seen at 0.2% galactose on the pGAL-BAX plate can be morphologically distinguished from the others, suggesting that they are contaminants.


Quantitative galactose induction assay

To further analyze the effect of BAX on our yeast under different expression levels, we conducted a quantitative galactose induction assay. Here, an O/N culture of yeast containing pGAL1-BAX was diluted to an OD600nm of 0.5. Subsequently, 100 µl of the culture in dilutions of 10-3 and 10-4 were spread on plates with 0%, 0.05%, 0.1%, 0.3% and 1% galactose, respectively. Each plate was made in duplicate. After incubation for three days at 30 °C, the CFU/ml were calculated and compared to the control (Figure 5).

[Discussion of below graph]

Ovulaid30.png

Figure 5: Gradient induction of BAX in yeast using the inducible GAL1 promoter. | If no errorbar is indicated, only one of the used duplets showed a quantifiable amount of colonies.

As seen on figure 6, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately our control strain was only plated on raff-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to pGAL1-BAX grown in 0 % galactose media). In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this that the cells were killed before a normal sized colony could be formed. To test this, the pGAL1-BAX plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed (Figure 6).

Ovulaid33.png

Figure 6: Comparison of colony sizes of pGAL1-BAX and the control containing the empty vector grown on raff-U agar with galactose. | Left: Colonies of the control strain (dilution 10-3) in the presence of 1% galactose after incubation for three days at 30 °C. Right: Colonies of the pGAL1-BAX strain (dilution 10-3) in the presence of 0.1% galactose after incubation for four days at 30 °C.

User Reviews

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