Difference between revisions of "Part:BBa K4365000"

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(→‎Characterization of the FIG1 promoter-driven expression)
 
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<partinfo>BBa_K4365000 short</partinfo>
 
<partinfo>BBa_K4365000 short</partinfo>
  
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The Factor-Induced Gene 1 (FIG1) is a pheromone-induced promoter in yeast that is activated by the alpha mating factor. The FIG1 promoter is a great device for synthetic biology applications aiming to engineer productive stationary-phase systems in S. cerevisiae. This is because the induction of the FIG1 promoter by the alpha mating factor, in addition to activating expression, leads to the arrest of growth and maintenance of active metabolism in S. cerevisiae. As a result, the synthesis of a product of interest is decoupled from population growth, and cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct. Moreover, the FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids cross-activation of other pathways and has enabled the construction and fine-tuning of a multitude of synthetic regulatory circuits.
+
The Factor-Induced Gene 1 (FIG1) promoter is a pheromone-induced promoter in yeast that is activated by the alpha mating factor. The FIG1 promoter could be used as a device to engineer productive stationary-phase systems in <i>Saccharomyces cerevisiae</i> and increase protein yield. Moreover, the pheromone response could be used for artificial cell-cell communication systems.
  
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<br>
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<b>Profile</b><br>
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Name: FIG1 promoter
 +
<br>Origin: <i>Saccharomyces cerevisiae</i>
 +
<br>Target organism: <i>Saccharomyces cerevisiae</i>
 +
<br>Main purpose of use: inducible heterologous protein expression
 +
<br>Notes: cultivate yeast in minimal media variants to avoid unregulated expression<br><br>
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4365000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4365000 SequenceAndFeatures</partinfo>
 
<br>
 
<br>
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 +
 
__TOC__
 
__TOC__
  
 
<br>
 
<br>
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===Biology and Usage===
 
===Biology and Usage===
 
====Factor-Induced Gene 1 and pheromone induced mating pathway====
 
====Factor-Induced Gene 1 and pheromone induced mating pathway====
−
<b>Factor-Induced Gene 1 (FIG1)</b> is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 <ref name="Pincus">Pincus D, Ryan CJ, Smith RD, Brent R, Resnekov O. Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output [published correction appears in PLoS One. 2013;8(6). doi: 10.1371/annotation/06dfa4e4-30f5-4d37-8559-0f2a9d11f0de]. PLoS One. 2013;8(3):e56544. doi:10.1371/journal.pone.0056544</ref>. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in <i>Saccharomyces cerevisiae</i>) binds to its receptor (Ste2) which leads to signal transduction via MAP kinase cascade. This results in phosphorylation of Ste12 transcription factor and allows it to bind to PRE motif present in pheromone-responsive genes promoter <ref name="Wong">Wong Sak Hoi J, Dumas B. Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell. 2010 Apr;9(4):480-5. doi: 10.1128/EC.00333-09. Epub 2010 Feb 5. PMID: 20139240; PMCID: PMC2863410.</ref>.
+
Factor-Induced Gene 1 (FIG1) is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 <ref name="Pincus">Pincus D, Ryan CJ, Smith RD, Brent R, Resnekov O. Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output [published correction appears in PLoS One. 2013;8(6). doi: 10.1371/annotation/06dfa4e4-30f5-4d37-8559-0f2a9d11f0de]. PLoS One. 2013;8(3):e56544. doi:10.1371/journal.pone.0056544</ref>. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in <i>Saccharomyces cerevisiae</i>) binds to its receptor (Ste2) which leads to signal transduction via MAP kinase cascade. This results in phosphorylation of Ste12 transcription factor and allows it to bind to PRE motif present in pheromone-responsive genes promoter <ref name="Wong">Wong Sak Hoi J, Dumas B. Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell. 2010 Apr;9(4):480-5. doi: 10.1128/EC.00333-09. Epub 2010 Feb 5. PMID: 20139240; PMCID: PMC2863410.</ref>.
 +
 
 +
[[File:BBa_K4365000_Fig1_fig1.png|thumb|<b>Figure 1.</b> Induction of turboRFP expression driven by the FIG1 promoter by addition of 500 nM alpha mating factor pheromone into the growth medium. The image was taken under a UV lamp.]]
  
 
====FIG1 promoter and its advantages for synthethic biology in yeast====
 
====FIG1 promoter and its advantages for synthethic biology in yeast====
−
 
−
[[File:BBa_K4365000_Fig1_fig1.png|thumb|<b>Figure 1.</b> Induction of turboRFP expression driven by the FIG1 promoter by addition of 500 nM alpha mating factor pheromone into the growth medium. The image was taken under a UV lamp.]]
 
  
 
FIG1 promoter is a pheromone-induced promoter that is activated by the alpha mating factor. This promoter can be applied for engineering productive stationary-phase systems in <i>S. cerevisiae</i> and has been used to improve heterologous protein yield <ref name="Thomas">Thomas C. Williams, Bingyin Peng, Claudia E. Vickers, Lars K. Nielsen, The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production, Metabolic Engineering Communications, Volume 3, 2016, Pages 142-152, ISSN 2214-0301, https://doi.org/10.1016/j.meteno.2016.05.001.</ref> or to control cell-cell communication in yeast cultures <ref>Hennig, S., Rödel, G. & Ostermann, K. Artificial cell-cell communication as an emerging tool in synthetic biology applications. J Biol Eng 9, 13 (2015). https://doi.org/10.1186/s13036-015-0011-2.</ref>. FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways <ref name="Thomas" />.
 
FIG1 promoter is a pheromone-induced promoter that is activated by the alpha mating factor. This promoter can be applied for engineering productive stationary-phase systems in <i>S. cerevisiae</i> and has been used to improve heterologous protein yield <ref name="Thomas">Thomas C. Williams, Bingyin Peng, Claudia E. Vickers, Lars K. Nielsen, The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production, Metabolic Engineering Communications, Volume 3, 2016, Pages 142-152, ISSN 2214-0301, https://doi.org/10.1016/j.meteno.2016.05.001.</ref> or to control cell-cell communication in yeast cultures <ref>Hennig, S., Rödel, G. & Ostermann, K. Artificial cell-cell communication as an emerging tool in synthetic biology applications. J Biol Eng 9, 13 (2015). https://doi.org/10.1186/s13036-015-0011-2.</ref>. FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways <ref name="Thomas" />.
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The induction of the FIG1 promoter by the alpha mating factor not only activates the expression of a gene of interest, but also stops the growth and maintenance of active metabolism in <i>S. cerevisiae</i>. As a result, the synthesis of a product of interest becomes independent of population growth. At the same time, cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct <ref name="Thomas" />.
 
The induction of the FIG1 promoter by the alpha mating factor not only activates the expression of a gene of interest, but also stops the growth and maintenance of active metabolism in <i>S. cerevisiae</i>. As a result, the synthesis of a product of interest becomes independent of population growth. At the same time, cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct <ref name="Thomas" />.
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==== References ====
 
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<references />
 
  
 
<br>
 
<br>
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===Characterization of FIG1 promoter===
 
===Characterization of FIG1 promoter===
 
====Pheromone response induction and activation of the FIG1 promoter in Δfar1 Δbar1 strain====
 
====Pheromone response induction and activation of the FIG1 promoter in Δfar1 Δbar1 strain====
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[[File:BBa K4365000 fig2.png|thumb|<b>Figure 2.</b> Growth curves of Δfar1Δbar1 <i>S. cerevisiae</i> cells after induction with the 500 nM of alpha mating factor pheromone.]]
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[[File:BBa K4365000 fig2.png|thumb|<b>Figure 2.</b> Growth curves of Δfar1Δbar1 <i>S. cerevisiae</i> cells after induction with the 500 nM of alpha mating factor pheromone. The data was plotted using R-studio.]]
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To characterize the *FIG1* promoter and the pheromone response it induces we conducted a series of tests. We first verified the effect of the alpha mating factor pheromone on growth by culturing yeast cells in two conditions in flasks. BY4741 yeast strain with Δfar1 and Δbar1 deletions (Euroscarf, Acc. No. Y00000). These deletions prevent the degradation of the pheromone [12] and the cell cycle arrest [13]. The far1 deletion is beneficial to sensing systems as it avoids complete arrest of the cell cycle so that the strain does not get lost.
+
  
−
The two yeast cultures were grown overnight in MV medium at 30°C in a shaking incubator. The following day, a 20 mL culture was prepared by adjusting the overnight cultures to 1 OD and 500 nM alpha mating factor pheromone were added into the medium to activate the pheromone response.  
+
To verify the effect of the alpha mating factor pheromone on growth, we cultured yeast cells in two conditions in flasks. BY4741 yeast strain with Δfar1 and Δbar1 deletions (Euroscarf, Acc. No. Y00000). These deletions prevent the degradation of the pheromone <ref name="Barkai">Barkai, N., Rose, M. & Wingreen, N. Protease helps yeast find mating partners. Nature 396, 422–423 (1998). https://doi.org/10.1038/24760 </ref> and the cell cycle arrest <ref name="Fred">Fred Chang, Ira Herskowitz, Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2, Cell, Volume 63, Issue 5, 1990, Pages 999-1011, ISSN 0092-8674, https://doi.org/10.1016/0092-8674(90)90503-7.</ref>. The far1 deletion is beneficial to sensing systems as it avoids complete arrest of the cell cycle so that the strain does not get lost.  
  
−
The growth of the cultures was monitored over the course of several hours, starting with 12 hours after induction, by measuring their optical density using a spectrophotometer (Figure 5). Due to the size of the yeast cells, it was always necessary to perform a 1:10 dilution of the yeast culture before measuring the OD. The data was plotted using R-studio.
+
The two yeast cultures were grown overnight in MV medium at 30°C in a shaking incubator. The following day, a 20 mL culture was prepared by adjusting the overnight cultures to 1 OD and 500 nM alpha mating factor pheromone were added into the medium to activate the pheromone response.  
  
−
The growth curves show that the pheromone is still able to slow down growth despite the deletion of the FAR1 gene.
+
The growth of the cultures was monitored over the course of several hours, starting with 12 hours after induction, by measuring their optical density using a spectrophotometer (Figure 2). Due to the size of the yeast cells, it was always necessary to perform a 1:10 dilution of the yeast culture before measuring the OD. The resulted growth curves show that the pheromone is still able to slow down growth despite the deletion of the FAR1 gene.
  
 
====Characterization of the FIG1 promoter-driven expression====
 
====Characterization of the FIG1 promoter-driven expression====
  
−
To assess the degree of leakiness in the regulation of the *FIG1* promoter we performed two sets of experiments.
+
To assess the degree of leakiness in the regulation of the FIG1 promoter we performed two sets of experiments.
  
−
We transformed the BY4741 ∆far1 ∆bar1 yeast strain with the p426 shuttle plasmid [14] for the expression of turboRFP under the control of the *FIG1* promoter. An overnight culture of this yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures, each at 1 OD. One of the two cultures was induced with 500 nM alpha mating factor while the other was left uninduced as a control. A sample of each culture was then spun down and imaged using both brightfield and TexasRed emission channels. This was repeated for a total of 5 time points, starting at 0 hours and running until 4 hours after induction.
+
We transformed the BY4741 ∆far1 ∆bar1 yeast strain with the p426 shuttle plasmid <ref name="mumberg">Mumberg D, Müller R, Funk M. (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds, Gene;156(1):119-122. doi:10.1016/0378-1119(95)00037-7</ref> for the expression of <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K4365020">turboRFP</a></html> under the control of the FIG1 promoter. An overnight culture of the yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures, each at 1 OD. One of the two cultures was induced with 500 nM alpha mating factor while the other was left uninduced as a control. A sample of each culture was then spun down and imaged using both brightfield and TexasRed emission channels. This was repeated for a total of 5 time points, starting at 0 hours and running until 4 hours after induction.
  
 
[[File:BBa K4365000 fig3.png|600px|center|thumb|<b>Figure 3.</b> Experiment of visualization of turboRFP expression driven by the FIG1 promoter in induced (500 nM alpha mating factor pheromone) and uninduced state. The images were taken in bright field mode and with the TexasRed emission channel using the Keyence Biozero fluorescence microscope.]]
 
[[File:BBa K4365000 fig3.png|600px|center|thumb|<b>Figure 3.</b> Experiment of visualization of turboRFP expression driven by the FIG1 promoter in induced (500 nM alpha mating factor pheromone) and uninduced state. The images were taken in bright field mode and with the TexasRed emission channel using the Keyence Biozero fluorescence microscope.]]
  
−
Next to no turboRFP-positive cells were observed in the control group while the signal was consistently present in the induced group and accumulated over time as is shown by the increase from time points taken at 2 and 4 hours. To get a more detailed analysis a follow-up experiment was conducted using flow cytometry.  
+
Next to no turboRFP-positive cells were observed in the control group while the signal was consistently present in the induced group and accumulated over time as is shown by the increase from time points taken at 2 and 4 hours (Figure 3). To get a more detailed analysis a follow-up experiment was conducted using flow cytometry.
  
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+
=====Single Cell Resolution using Cyflow=====
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====Single Cell Resolution using Cyflow====
+
We transformed yeast with the p426 shuttle plasmid <ref name="mumberg" /> for the expression of EGFP under the control of the FIG1 promoter.  In this experiment, we also tested the influence of rich (MV) and minimal (W0) medium on the regulation of the FIG1 promoter.
−
To further demonstrate the consistency and potency of induction of the *FIG1* via the alpha mating factor pheromone, we transformed yeast with the p426 shuttle plasmid [14] for the expression of EGFP under the control of the *FIG1* promoter.  In this experiment, we also tested the influence of rich (MV) and minimal (W0) medium on the regulation of the *FIG1* promoter.
+
  
 
An overnight culture of this yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures in rich MV medium and minimal W0. All four cultures were adjusted to an OD of 1. One of the cultures in MV and one of the cultures in W0 was induced with 500 nM alpha mating factor while the other was left noninduced as a control. The cultures were grown at 30°C in a shaking incubator.
 
An overnight culture of this yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures in rich MV medium and minimal W0. All four cultures were adjusted to an OD of 1. One of the cultures in MV and one of the cultures in W0 was induced with 500 nM alpha mating factor while the other was left noninduced as a control. The cultures were grown at 30°C in a shaking incubator.
  
−
Induced and non-induced cell populations were analyzed by cytometry to determine the EGFP intensity per cell after 1 hour and 3.5 hours after the inoculation of the alpha mating factor pheromone into the culture. For each analysis 20,000 total cells were measured. Analysis of the cyflow data was conducted using online tools at [floreada.io](https://floreada.io/). The experimental groups and plots of the obtained cyflow results are summarized below.
+
Induced and non-induced cell populations were analyzed by cytometry to determine the EGFP intensity per cell after 1 hour and 3.5 hours after the inoculation of the alpha mating factor pheromone into the culture. For each analysis 20,000 total cells were measured. Analysis of the cyflow data was conducted using online tools at <html><a href="http://floreada.io/">floreada.io</a></html>. The experimental groups and plots of the obtained cyflow results are summarized below.
  
  
Line 77: Line 79:
  
  
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From these results, it is possible to observe that the induction of the FIG1 promoter is well regulated and exhibits minimal leakiness when in the W0 medium conditions. The mean intensity for the non-induced population was found to be 9.5 at 1 hour and 8.59 at 3.5 hours. Seeing as the mean does not increase over time this signal can likely be explained as background signal during measurement, not originating from any gene expression. For the induced sample in W0 medium the mean intensity was found to be 15.91 at 1 hour and 51.89 at 3.5 hours showing a dramatic increase over time. However, in the MV media condition the measured intensity was found to increase for both induced and non-induced conditions over time. The non-induced sample increased from a mean intensity of 8.8 at 1 hour to 10.46 at 3.5 hours, at the same timepoints the induced sample increased from 15.12 to 52.43. This slight shift in non-induced intensity can be appreciated in figure 8.C-D (blue). This increase can also be seen between media conditions, where the non-induced control in MV appears to slightly gain fluorescence (Figure 8, blue, D) compared to the same population grown in minimal medium (Figure 8, blue, B). It seems that the use of rich medium (MV) may have an effect on the induction of the FIG1 promotor causing aberrant gene expression in the non-induced samples. It is therefore possible that MV media increases the overall expression regardless of the presence of the alpha mating factor pheromone. We therefore recommend that yeast be cultivated in minimal media variants to avoid unregulated expression of the target protein when using the FIG1 promoter.
+
From these results, it is possible to observe that the induction of the FIG1 promoter is well regulated and exhibits minimal leakiness when in the W0 medium conditions. The mean intensity for the non-induced population was found to be 9.5 at 1 hour and 8.59 at 3.5 hours. Seeing as the mean does not increase over time this signal can likely be explained as background signal during measurement, not originating from any gene expression. For the induced sample in W0 medium the mean intensity was found to be 15.91 at 1 hour and 51.89 at 3.5 hours showing a dramatic increase over time. However, in the MV media condition the measured intensity was found to increase for both induced and non-induced conditions over time. The non-induced sample increased from a mean intensity of 8.8 at 1 hour to 10.46 at 3.5 hours, at the same timepoints the induced sample increased from 15.12 to 52.43. This slight shift in non-induced intensity can be appreciated in Figure 4.C-D (blue). This increase can also be seen between media conditions, where the non-induced control in MV appears to slightly gain fluorescence (Figure 4, blue, D) compared to the same population grown in minimal medium (Figure 4, blue, B). It seems that the use of rich medium (MV) may have an effect on the induction of the FIG1 promotor causing aberrant gene expression in the non-induced samples. It is therefore possible that MV media increases the overall expression regardless of the presence of the alpha mating factor pheromone.  
  
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We therefore recommend that yeast be <b>cultivated in minimal media variants</b> to avoid unregulated expression of the target protein when using the FIG1 promoter.
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=== References ===
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<references />
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4365000 parameters</partinfo>
 
<partinfo>BBa_K4365000 parameters</partinfo>
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Latest revision as of 08:35, 12 October 2022


FIG1 inducible promoter

The Factor-Induced Gene 1 (FIG1) promoter is a pheromone-induced promoter in yeast that is activated by the alpha mating factor. The FIG1 promoter could be used as a device to engineer productive stationary-phase systems in Saccharomyces cerevisiae and increase protein yield. Moreover, the pheromone response could be used for artificial cell-cell communication systems.

Profile
Name: FIG1 promoter
Origin: Saccharomyces cerevisiae
Target organism: Saccharomyces cerevisiae
Main purpose of use: inducible heterologous protein expression
Notes: cultivate yeast in minimal media variants to avoid unregulated expression

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 172
  • 1000
    COMPATIBLE WITH RFC[1000]




Biology and Usage

Factor-Induced Gene 1 and pheromone induced mating pathway

Factor-Induced Gene 1 (FIG1) is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 [1]. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in Saccharomyces cerevisiae) binds to its receptor (Ste2) which leads to signal transduction via MAP kinase cascade. This results in phosphorylation of Ste12 transcription factor and allows it to bind to PRE motif present in pheromone-responsive genes promoter [2].

Figure 1. Induction of turboRFP expression driven by the FIG1 promoter by addition of 500 nM alpha mating factor pheromone into the growth medium. The image was taken under a UV lamp.

FIG1 promoter and its advantages for synthethic biology in yeast

FIG1 promoter is a pheromone-induced promoter that is activated by the alpha mating factor. This promoter can be applied for engineering productive stationary-phase systems in S. cerevisiae and has been used to improve heterologous protein yield [3] or to control cell-cell communication in yeast cultures [4]. FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways [3].

The induction of the FIG1 promoter by the alpha mating factor not only activates the expression of a gene of interest, but also stops the growth and maintenance of active metabolism in S. cerevisiae. As a result, the synthesis of a product of interest becomes independent of population growth. At the same time, cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct [3].


Characterization of FIG1 promoter

Pheromone response induction and activation of the FIG1 promoter in Δfar1 Δbar1 strain

Figure 2. Growth curves of Δfar1Δbar1 S. cerevisiae cells after induction with the 500 nM of alpha mating factor pheromone. The data was plotted using R-studio.

To verify the effect of the alpha mating factor pheromone on growth, we cultured yeast cells in two conditions in flasks. BY4741 yeast strain with Δfar1 and Δbar1 deletions (Euroscarf, Acc. No. Y00000). These deletions prevent the degradation of the pheromone [5] and the cell cycle arrest [6]. The far1 deletion is beneficial to sensing systems as it avoids complete arrest of the cell cycle so that the strain does not get lost.

The two yeast cultures were grown overnight in MV medium at 30°C in a shaking incubator. The following day, a 20 mL culture was prepared by adjusting the overnight cultures to 1 OD and 500 nM alpha mating factor pheromone were added into the medium to activate the pheromone response.

The growth of the cultures was monitored over the course of several hours, starting with 12 hours after induction, by measuring their optical density using a spectrophotometer (Figure 2). Due to the size of the yeast cells, it was always necessary to perform a 1:10 dilution of the yeast culture before measuring the OD. The resulted growth curves show that the pheromone is still able to slow down growth despite the deletion of the FAR1 gene.

Characterization of the FIG1 promoter-driven expression

To assess the degree of leakiness in the regulation of the FIG1 promoter we performed two sets of experiments.

We transformed the BY4741 ∆far1 ∆bar1 yeast strain with the p426 shuttle plasmid [7] for the expression of turboRFP under the control of the FIG1 promoter. An overnight culture of the yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures, each at 1 OD. One of the two cultures was induced with 500 nM alpha mating factor while the other was left uninduced as a control. A sample of each culture was then spun down and imaged using both brightfield and TexasRed emission channels. This was repeated for a total of 5 time points, starting at 0 hours and running until 4 hours after induction.

Figure 3. Experiment of visualization of turboRFP expression driven by the FIG1 promoter in induced (500 nM alpha mating factor pheromone) and uninduced state. The images were taken in bright field mode and with the TexasRed emission channel using the Keyence Biozero fluorescence microscope.

Next to no turboRFP-positive cells were observed in the control group while the signal was consistently present in the induced group and accumulated over time as is shown by the increase from time points taken at 2 and 4 hours (Figure 3). To get a more detailed analysis a follow-up experiment was conducted using flow cytometry.

Single Cell Resolution using Cyflow

We transformed yeast with the p426 shuttle plasmid [7] for the expression of EGFP under the control of the FIG1 promoter. In this experiment, we also tested the influence of rich (MV) and minimal (W0) medium on the regulation of the FIG1 promoter.

An overnight culture of this yeast strain was prepared in W0 minimal medium and the next day it was split to make two main cultures in rich MV medium and minimal W0. All four cultures were adjusted to an OD of 1. One of the cultures in MV and one of the cultures in W0 was induced with 500 nM alpha mating factor while the other was left noninduced as a control. The cultures were grown at 30°C in a shaking incubator.

Induced and non-induced cell populations were analyzed by cytometry to determine the EGFP intensity per cell after 1 hour and 3.5 hours after the inoculation of the alpha mating factor pheromone into the culture. For each analysis 20,000 total cells were measured. Analysis of the cyflow data was conducted using online tools at floreada.io. The experimental groups and plots of the obtained cyflow results are summarized below.


Yeast Medium Impacts Expression tightness of the FIG1 promoter
+ Alpha Factor - Alpha Factor
MV Medium MV induced MV uninduced
W0 Medium W0 induced W0 uninduced
Figure 4. Cytoflow results for EGFP intensity in induced and non-induced cultures. A: W0 medium sample at 1 hour, induced (green) non-induced (blue). A: W0 medium sample after 3.5 hours, induced (green) non-induced (blue). C: MV medium sample at 1 hour, induced (green) non-induced (blue). D: MV medium sample at 3.5 hours, induced (green) non-induced (blue). The fluorescence of 20 000 cells was measured.


From these results, it is possible to observe that the induction of the FIG1 promoter is well regulated and exhibits minimal leakiness when in the W0 medium conditions. The mean intensity for the non-induced population was found to be 9.5 at 1 hour and 8.59 at 3.5 hours. Seeing as the mean does not increase over time this signal can likely be explained as background signal during measurement, not originating from any gene expression. For the induced sample in W0 medium the mean intensity was found to be 15.91 at 1 hour and 51.89 at 3.5 hours showing a dramatic increase over time. However, in the MV media condition the measured intensity was found to increase for both induced and non-induced conditions over time. The non-induced sample increased from a mean intensity of 8.8 at 1 hour to 10.46 at 3.5 hours, at the same timepoints the induced sample increased from 15.12 to 52.43. This slight shift in non-induced intensity can be appreciated in Figure 4.C-D (blue). This increase can also be seen between media conditions, where the non-induced control in MV appears to slightly gain fluorescence (Figure 4, blue, D) compared to the same population grown in minimal medium (Figure 4, blue, B). It seems that the use of rich medium (MV) may have an effect on the induction of the FIG1 promotor causing aberrant gene expression in the non-induced samples. It is therefore possible that MV media increases the overall expression regardless of the presence of the alpha mating factor pheromone.

We therefore recommend that yeast be cultivated in minimal media variants to avoid unregulated expression of the target protein when using the FIG1 promoter.

References

  1. ↑ Pincus D, Ryan CJ, Smith RD, Brent R, Resnekov O. Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output [published correction appears in PLoS One. 2013;8(6). doi: 10.1371/annotation/06dfa4e4-30f5-4d37-8559-0f2a9d11f0de]. PLoS One. 2013;8(3):e56544. doi:10.1371/journal.pone.0056544
  2. ↑ Wong Sak Hoi J, Dumas B. Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell. 2010 Apr;9(4):480-5. doi: 10.1128/EC.00333-09. Epub 2010 Feb 5. PMID: 20139240; PMCID: PMC2863410.
  3. ↑ 3.0 3.1 3.2 Thomas C. Williams, Bingyin Peng, Claudia E. Vickers, Lars K. Nielsen, The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production, Metabolic Engineering Communications, Volume 3, 2016, Pages 142-152, ISSN 2214-0301, https://doi.org/10.1016/j.meteno.2016.05.001.
  4. ↑ Hennig, S., Rödel, G. & Ostermann, K. Artificial cell-cell communication as an emerging tool in synthetic biology applications. J Biol Eng 9, 13 (2015). https://doi.org/10.1186/s13036-015-0011-2.
  5. ↑ Barkai, N., Rose, M. & Wingreen, N. Protease helps yeast find mating partners. Nature 396, 422–423 (1998). https://doi.org/10.1038/24760
  6. ↑ Fred Chang, Ira Herskowitz, Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2, Cell, Volume 63, Issue 5, 1990, Pages 999-1011, ISSN 0092-8674, https://doi.org/10.1016/0092-8674(90)90503-7.
  7. ↑ 7.0 7.1 Mumberg D, Müller R, Funk M. (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds, Gene;156(1):119-122. doi:10.1016/0378-1119(95)00037-7