Difference between revisions of "Part:BBa K2587009"

 
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<partinfo>BBa_K2587009 short</partinfo>
 
<partinfo>BBa_K2587009 short</partinfo>
  
<p>Most of the microorganisms, especially common used model organsims like <i>Escherichia coli</i> or <i>Saccharomyces cerevisiae</i> grow only very slow on phosphite as phosphorus source. Moreover in industry is not widely accepted to use antibiotic resistances as markers. Therefore a system is required, which allows avoidance of contamination by other microorganisms and at the same time represents a reliable selection marker. We present the use of the ptxD gene from <i>Pseudomonas stutzeri</i> together with a <strong>phosphite media</strong> (reduces growth of contaminants), which could abolish the use of antibiotics in the future and the more efficient use of a phosphorus source in the medium. In this case ptxD is codon optimised for <i>S.cerevisiae</i><sup>1</sup>.</p>
+
<p><i>ptxD</i> allows microorganisms to start metabolizing phosphite, also known as phosphonic acid, an alternative phosphorus source not commonly metabolizable by most organisms.
 +
In industry applications, the use of antibiotic resistnace markers is not widely accepted. Therefore, a system which avoids contamination by other microorganisms in a different manner, while still functioning as a reliable selection marker, is required. We present the use of the <i>ptxD</i> gene from <i>Pseudomonas stutzeri</i> together with <strong>phosphite media</strong> (which reduces growth of contaminants). This could abolish the use of antibiotics. In this case, <i>ptxD</i> is codon optimized for <i>S.cerevisiae</i><sup>1</sup>.</p>
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<br>
 
<br>
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<p><ul>
 
<p><ul>
<li> ptxD codes for phosphonate dehydrogenase</li>
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<li> <i>ptxD</i> encodes phosphonate dehydrogenase</li>
<li> oxidation of phosphite (phosphonate) using NAD+ and H<sub>2</sub>0 to phosphate and NADH </li>
+
<li> Oxidation of phosphite (phosphonate) using NAD<sub>+</sub> and H<sub>2</sub>O to phosphate and NADH </li>
 
<li> Selection marker for budding and fission yeast</li>
 
<li> Selection marker for budding and fission yeast</li>
<li> phosphite-oxidizing ability </li>
+
<li> Phosphite-oxidizing ability </li>
<li> environmentally safe culture</li>  
+
<li> Environmentally safe culture</li>  
<li> antibiotic free system</li>
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<li> Antibiotic free system</li>
<li> pH optimum: 7.25- 7.75</li>
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<li> pH optimum: 7.25 - 7.75</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
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<p>
 
<p>
 
<strong><h5>Experimental Design</h5></strong>
 
<strong><h5>Experimental Design</h5></strong>
In our three-way co-culture we want to use phosphite as a non-metabolizable phosphorus source. Only our engineered <i>S.&nbsp; cerevisiae</i> strain is able to convert it to phosphate for itself, as well as providing it to the other organisms.
+
In our three-way co-culture, we want to use phosphite as a non-metabolizable phosphorus source. Only our engineered <i>S.cerevisiae</i> strain is able to convert it to phosphate for itself, as well as providing it to the other organisms.
To test if our construct with the codon optimized <i>ptxD</i> gene (<i>ptxD</i>_opt)<sup>1</sup> works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. <i>S. cerevisiae</i> and <i>E. coli</i> , both used as negative control, were cultivated in standard M2 medium<sup>2</sup> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid. The same supplements were used to make the media only differ in the phosphorus source. Medium lacking uracil was used in the samples containing our construct, to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. As sample size 200 µl were chosen. Every time, OD<sub>600</sub> and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.<br></p>  
+
To test if our construct with the codon optimized <i>ptxD</i> gene (<i>ptxD</i>_opt)<sup>1</sup> works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. <i>S. cerevisiae</i> and <i>E. coli</i>, both used as negative control, were cultivated in standard M2 medium<sup>2</sup> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid. The same supplements were used to make the media differ only in the phosphorus source. Medium lacking uracil was used in the samples containing our construct, to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. As sample size 200 µl were chosen. Every time, OD<sub>600</sub> and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.<br></p>  
 
<br>
 
<br>
<p><strong>Table 2: Loading scheme of the 96 well plate for the OD measurement of different cultures, different colors represent different media compositions:</strong></p>
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<p><strong>Table 2: Loading scheme of the 96 well plate for the OD<sub>600</sub> measurement of different cultures, different colors represent different media compositions:</strong></p>
https://static.igem.org/mediawiki/parts/a/a7/T--Duesseldorf--loading_scheme_96_well_plate.JPG
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https://static.igem.org/mediawiki/parts/2/20/T--Duesseldorf--loading_scheme_96_well_plate_besser.JPG
 +
https://static.igem.org/mediawiki/parts/2/29/T--Duesseldorf--loading_scheme_96_well_plate_besser_legende_klein.JPG
 +
<p><strong><h5>Data</h5></strong></p>
  
<p><strong><h5>Data</h5></strong></p><br>
+
<p>First it had to be tested whether other organisms in the co-culture were able to use phosphite as a phosphorus source. To test this, we compared growth of <i>E. coli</i> and <i>S. cerevisiae</i> in normal M2 medium with M2 medium where phosphite was the only phosphorus source.<br></p>
  
<p>First it had to be tested whether other organisms in the co-culture are able to use phosphite as a phosphorus source. To test this, we compared growth of <i>E. coli</i> and <i>S. cerevisiae</i> in normal M2 medium with M2 medium where phosphite is the only phosphorus source.<br></p>
+
https://static.igem.org/mediawiki/parts/f/f1/T--Duesseldorf--Result3.Fig1standartabweichungklein.png
 +
<p><strong>Figure 1: Growth of <i>E. coli</i> over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but only with phosphite instead of phosphate (yellow). The cell density was measured using OD<sub>600</sub>.</p>
  
<img class=half-width scr="https://static.igem.org/mediawiki/2018/b/b1/T--Duesseldorf--Result3.Fig4.png">
+
<p>As shown in Figure 1, <i>E.&nbsp;coli</i> shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours in standard M2 medium. After 20 hours,  <i>E.coli</i> reaches a plateau with an OD<sub>600</sub> of nearly 0.4. In M2 medium with phosphite, the bacteria stay in the lag phase and only reach an OD<sub>600</sub> of less than 0.1. At the end of the measurement a slow decrease of the population is visible.</p>
 +
<br>
 +
https://static.igem.org/mediawiki/parts/4/44/T--Duesseldorf--Result3.Fig2standartabweichungklein.png
 +
<p><strong>Figure 2: Growth of <i>S. cerevisiae</i> over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine&nbsp;(1.52&nbsp;mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but containing phosphite instead of phosphate (yellow), measured with OD<sub>600</sub>.</p>
  
<p><strong>Figure 1: Growth of <i>E. coli</i> in different medium over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but only with phosphite instead of phosphate (yellow). The cell density was measured using OD<sub>600</sub>.</p>
+
<p><i>S. cerevisiae</i> shows a growth curve similar to <i>E. coli</i>, with a lag phase up until 10 hours and a stationary phase after 20 hours (Figure 2). For cells incubated in M2 medium, supplemented with phosphite as the sole phosphorus source, no growth is detectable.<br></p>
 
<br>
 
<br>
<p>As shown in Figure 1 in standard M2 medium <i>E.&nbsp;coli</i> shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours. After 20 hours  <i>E.&nbsp;coli</i> reaches the stationary phase with an OD<sub>600</sub> of nearly 0.4. In M2 medium with phosphite the bacteria stay in the lag phase and only reach an OD<sub>600</sub> of less than 0.1. At the end of the measurement a slow decrease of the population is visible.</p>
+
<p>To figure out the strongest one, different promoters from the YTK toolbox<sup>3</sup> were tested: TDH3 (BBa_K124002, Link: https://parts.igem.org/Part:BBa_K124002), CCW12, PGK1 (BBa_K122000, Link:https://parts.igem.org/Part:BBa_K122000), HHF2, TEF1 controlling the <i>ptxD</i>_opt gene in <i>S. cerevisiae</i>. All previously described experiments were also performed using M2 medium with phosphite as phosphorus source.</p>  
<br>
+
https://static.igem.org/mediawiki/parts/8/87/T--Duesseldorf--Result3.Fig3klein.png
<p><strong>Figure 2: Growth of <i>S. cerevisiae</i> over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine&nbsp;(1.52&nbsp;mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but containing phosphite instead of phosphate (yellow), measured with OD<sub>600</sub>.<br></p>
+
<p><strong>Figure 3:  Growth of five <i>S. cerevisiae</i> strains containing <i>ptxD</i>_opt under the control of different promoters over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite. Strains with promoter TDH3 (blue), CCW12 (red), PGK1 (green), HHF2 (gray), TEF1 (yellow), measured at OD<sub>600</sub>.</p>
 
+
<p>All constructs with the five promoters show a constant, but slightly different growth in M2 medium containing phosphite (Figure 3). With the strongest promoter TDH3, <i>S. cerevisiae</i> achieves an OD<sub>600</sub> of nearly 0.025, which is more than 100% more growth than the weakest promoter of the five: CCW12. The strains with the promoters PGK1 or TEF1 reach an OD<sub>600</sub> of a little bit over 0.015, representing medium strength.</p><br>
<p><i>S. cerevisiae</i> shows the common growth curve similar to <i>E. coli</i>, with a lag phase until 10 hours and a stationary phase after 20 hours (Figure 2). Cells incubated in M2 medium, supplemented with phosphite as sole phosphorus source, no growth is detectable.<br></p>
+
<p>The final question is if our modified <i>S. cerevisiae</i> strains show a better growth on phosphite than the progenitor <i>S. cerevisiae</i>. Therefore, the growth of the strain with the <i>ptxD</i>_opt gene and the strongest promoter TDH3 was compared with the progenitor strain.</p>
 
+
https://static.igem.org/mediawiki/parts/6/6c/T--Duesseldorf--Result3.Fig4standartabweichungklein.png
<p>To figure out the strongest one, different promoters from the YTK toolbox<sup>3</sup> were tested: TDH3 (BBa_K124002, Link: https://parts.igem.org/Part:BBa_K124002), CCW12, PGK1 (BBa_K122000, Link:https://parts.igem.org/Part:BBa_K122000), HHF2, TEF1 controlling the <i>ptxD</i>_opt gene in <i>S. cerevisiae</i>. All previously described experiments were also performed using M2 medium with phosphite as phosphorus source.<br></p>  
+
<p><strong>Figure 4: Growth of the <i>S. cerevisiae</i> control strain (yellow) and <i>S. cerevisiae</i> with the <i>ptxD</i>_opt and TDH3 (blue) on M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and  lysine (1.52 mg/l) with phosphite over 52 h, measured at 600 nm. For the <i>S. cerevisiae</i> BY4742 background strain, uracil (18 mg/l) was added.</p>
<p><strong>Figure 3:  Growth of five <i>S. cerevisiae</i> strains with the <i>ptxD</i>_opt under different promoter over 52 h in M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite. Strain with promoter TDH3 (blue), CCW12 (red), PGK1 (green), HHF2 (gray), TEF1 (yellow), measured with OD<sub>600</sub>.</p>
+
<p>In Figure 4, the graph shows an initial growth of the progenitor strain <i>S. cerevisiae</i>, but the growth stops at an OD<sub>600</sub> of less than 0.03 after 10 hours. After 10&nbsp;hours, the  cell density of <i>S. cerevisiae</i> starts to decrease, slowly in the beginning, but faster towards the end (around 50 h). At the end of the experiment the progenitor strain has an OD<sub>600</sub> of less than 0.02.
<p>All constructs with the five promoters show a constant, but different growth in M2 medium with phosphite (Figure 3). With the strongest promoter TDH3 <i>S. cerevisiae</i> achieves an OD<sub>600</sub> of nearly 0.025, which is more than 100% more growth than the weakest promoter of the five: CCW12. The strains with the promoters PGK1 or TEF1 reach an OD<sub>600</sub> of a little bit over 0.015 and have middling strengths.</p>
+
The modified <i>S. cerevisiae</i> strain with <i>ptxD</i>_opt under the control of the TDH3 promoter starts at nearly the same OD<sub>600</sub> as the progenitor strain but after a short lag phase of around 5 hours, the strain grows, slowly in the beginning and faster towards the end, where it reaches an OD<sub>600</sub> of over 0.02.  
<p>The final question is if our modified <i>S. cerevisiae</i> strains shows a better growth on phosphite than the progenitor <i>S. cerevisiae</i>. Therefore, the growth of the strain with the <i>ptxD</i>_opt gene and the strongest promoter TDH3 were compared with the progenitor strain.</p>
+
Both strains start at the same OD<sub>600</sub> level. The non-modified strain grows faster than the modified strain at the beginning, but then decreases more and more, while the modified strain needs some time but shows a slow increase of growth afterwards.</p><br>
<p><strong>Figure 4: Growth of <i>S. cerevisiae</i> (blue) and <i>S. cerevisiae</i> with the <i>ptxD</i>_opt and TDH3 (yellow) on M2 medium</strong> with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and  lysine (1.52 mg/l) with phosphite over 52 h, measured with OD<sub>600</sub>. For the <i>S. cerevisiae</i> BY4742 background strain, uracil (18 mg/l) were added.</p>
+
<p><strong><h5>Conclusion</h5></strong></p>
<p>In Figure 4 the graph demonstrates an initial growth of the progenitor strain <i>S. cerevisiae</i>, but the growth stops at an OD<sub>600</sub> of less than 0.03 after 10 hours. After 10&nbsp;hours <i>S. cerevisiae</i> starts to decrease, in the beginning slowly, but in the end after over 50 hours a faster decrease is recognisable. At the end of the experiment the progenitor strain has an OD<sub>600</sub> of less than 0.02.  
+
<p>Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro nutrient essential for microorganisms. Creating a dependency based on the ability of <i>S. cerevisiae</i> to utilize an otherwise unusable phosphorus source like phosphite by expressing <i>ptxD</i> and making it metabolically available for <i>E. coli</i> is therefore a very promising approach.<br>
The modified <i>S. cerevisiae</i> strain with <i>ptxD</i>_opt and TDH3 starts nearly at the same OD<sub>600</sub> as the progenitor strain but after a short lag phase of around 5 hours, the strain grows, slowly in the beginning and a bit faster in the end, where it reaches an OD<sub>600</sub> of over 0.02.  
+
In order to build a stable dependency, the other organisms in this system have to lack the enzymes for the catalysis from phosphite to phosphorus. The experiment with <i>E. coli</i> (Figure 1) and <i>S. cerevisiae</i> (Figure 2) demonstrates that neither <i>E. coli</i> nor <i>S. cerevisiae</i> show any growth in M2 medium with phosphite. Figure 3 shows the growth of engineered <i>S. cerevisiae</i> with different promoter_<i>ptxD</i>_opt constructs. All show growth regardless of what promoter was used but the promoter TDH3 showed the strongest growth rates. This indicates that our approach can work. <br>
Both strains start at the same OD<sub>600</sub> level. The not modified strain grows initially faster than the modified strain, but then decreases more and more, while the modified strain needs some time but then shows a slowly rise of growth.</p>
+
When comparing the growth of both <i>S. cerevisiae</i> strains (Figure 4), some differences can be detected. The non-modified <i>S. cerevisiae</i> grows much faster in the beginning but soon thereafter begins to die. The modified strain however shows slow but constant growth. As a consequence, we assume that the modified strain would outcompete contaminating microorganisms and possibly the other members of the co-culture. To add to that, we have designed our co-culture with more than one dependency, for example with nitrogen or carbon.<br>
<p><strong><h5>Conclusion</h5></strong></p><br>
+
In general the growth was minimal, so for further experiments, higher concentrations of nutrients and phosphite might lead to better results. The slow growth rate of the modified strain is as we expected, because it is comparable to what the literature shows. There, a <i>ptxD</i> construct with the TEF1 promoter in <i>S. cerevisiae</i> was used and growth was monitored over 40 hours<sup>4</sup>. A constant temperature of 30°C also might increase growth. Moreover, a longer measurement time would also show the behavior of the culture over a longer time. This would be interesting because we would like to create a stable culture which can be maintained for as long as possible. In addition, co-culture experiments would likely lead to other results than monocultures. In this case it would be interesting to perform them as well with the same experimental design. For a future application in the co-culture, we suggest to use the phosphate exporter XPR1 from <i>Homo sapiens</i><sup>5,6</sup>. It may help to provide for the other organisms, due to the export of the produced phosphate.</p>
<p>Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro element essential for microorganisms. Creating a dependency based on the ability of <i>S. cerevisiae</i> to utilise an otherwise unusable phosphorus source like phosphite by expressing <i>ptxD</i> and making it metabolically available for <i>E. coli</i> is therefore a very promising approach.
+
<br>
In order to build a stable dependency the other organisms in this system have to lack the enzymes for the catalysis from phosphite to phosphorus. The experiment with <i>E. coli</i> (Figure 1) and <i>S. cerevisiae</i> (Figure 2) demonstrates that neither <i>E. coli</i> nor <i>S. cerevisiae</i> show any growth in M2 medium with phosphite. Figure 3 shows the growth of engineered <i>S. cerevisiae</i> with different promoter_<i>ptxD</i>_opt constructs. All show growth regardless of what promoter was used but the promoter TDH3 showed the strongest growth rates. This indicates that our approach can work.  
+
When comparing the growth of both <i>S. cerevisiae</i> strains (Figure 4), some differences can be detected. The non-modified <i>S. cerevisiae</i> grows much faster in the beginning but thereafter begins to die. The modified strain however shows slow, but constant growth. As a consequence, we assume that the modified strain would outcompete contaminating microorganisms and possibly the other members of the co-culture. But for that reason there are more than one dependency in the co-culture, for example with nitrogen.
+
In general the growth was minimal, so for further experiments, higher concentrations of nutrients and phosphite might lead to better results. The slow growth rate of the modified strain is as we expected, because it is comparable to what the literature shows. There, a <i>ptxD</i> construct with the TEF1 promoter in <i>S. cerevisiae</i> was used and growth was monitored over 40 hours<sup>4</sup>. A constant temperature of 30°C also might increase growth. Moreover, a longer measurement time could also show the behavior of the culture over a longer time. This would be interesting because we would like to create a stable culture which can be maintained as long as possible. In addition, co-culture experiments could lead to other results than monocultures. In this case it would be interesting to perform them as well with the same experimental design. For a future application in the co-culture, we suggest to use the phosphate exporter XPR1 from <i>Homo sapiens</i><sup>5,6</sup>. It may help to feed the other organisms, due to the secretion of the produced phosphate.</p>
+
+
 
<p><h4><strong>References</strong></h4></p>
 
<p><h4><strong>References</strong></h4></p>
  

Latest revision as of 23:49, 17 October 2018


ptxD_opt

ptxD allows microorganisms to start metabolizing phosphite, also known as phosphonic acid, an alternative phosphorus source not commonly metabolizable by most organisms. In industry applications, the use of antibiotic resistnace markers is not widely accepted. Therefore, a system which avoids contamination by other microorganisms in a different manner, while still functioning as a reliable selection marker, is required. We present the use of the ptxD gene from Pseudomonas stutzeri together with phosphite media (which reduces growth of contaminants). This could abolish the use of antibiotics. In this case, ptxD is codon optimized for S.cerevisiae1.



Usage and Biology

  • ptxD encodes phosphonate dehydrogenase
  • Oxidation of phosphite (phosphonate) using NAD+ and H2O to phosphate and NADH
  • Selection marker for budding and fission yeast
  • Phosphite-oxidizing ability
  • Environmentally safe culture
  • Antibiotic free system
  • pH optimum: 7.25 - 7.75

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 6
    Illegal BamHI site found at 1044
    Illegal XhoI site found at 216
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 714
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 22
    Illegal BsaI.rc site found at 1051


Results Phosphite Measurement


Experimental Design

In our three-way co-culture, we want to use phosphite as a non-metabolizable phosphorus source. Only our engineered S.cerevisiae strain is able to convert it to phosphate for itself, as well as providing it to the other organisms.

To test if our construct with the codon optimized ptxD gene (ptxD_opt)1 works, we performed a plate reader experiment over 52 hours with different M2 media characteristics. S. cerevisiae and E. coli, both used as negative control, were cultivated in standard M2 medium2 with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l). For some experiments, M2 medium was modified to contain phosphite (also known as phosphonic acid) instead of the originally used phosphoric acid. The same supplements were used to make the media differ only in the phosphorus source. Medium lacking uracil was used in the samples containing our construct, to maintain selection pressure. Five different constitutive promoters were tested. All samples were measured every 30 minutes in replicates of five. As sample size 200 µl were chosen. Every time, OD600 and temperature were measured. The experiment was performed at room temperature, while the plates were shaken vigorously.


Table 2: Loading scheme of the 96 well plate for the OD600 measurement of different cultures, different colors represent different media compositions:

T--Duesseldorf--loading_scheme_96_well_plate_besser.JPG T--Duesseldorf--loading_scheme_96_well_plate_besser_legende_klein.JPG

Data

First it had to be tested whether other organisms in the co-culture were able to use phosphite as a phosphorus source. To test this, we compared growth of E. coli and S. cerevisiae in normal M2 medium with M2 medium where phosphite was the only phosphorus source.

T--Duesseldorf--Result3.Fig1standartabweichungklein.png

Figure 1: Growth of E. coli over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but only with phosphite instead of phosphate (yellow). The cell density was measured using OD600.

As shown in Figure 1, E. coli shows the common growth curve with a lag phase of 10 hours and a log phase over 10 hours in standard M2 medium. After 20 hours, E.coli reaches a plateau with an OD600 of nearly 0.4. In M2 medium with phosphite, the bacteria stay in the lag phase and only reach an OD600 of less than 0.1. At the end of the measurement a slow decrease of the population is visible.


T--Duesseldorf--Result3.Fig2standartabweichungklein.png

Figure 2: Growth of S. cerevisiae over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l), lysine (1.52 mg/l) and uracil (18 mg/l) (blue) and in M2 medium with the same composition but containing phosphite instead of phosphate (yellow), measured with OD600.

S. cerevisiae shows a growth curve similar to E. coli, with a lag phase up until 10 hours and a stationary phase after 20 hours (Figure 2). For cells incubated in M2 medium, supplemented with phosphite as the sole phosphorus source, no growth is detectable.


To figure out the strongest one, different promoters from the YTK toolbox3 were tested: TDH3 (BBa_K124002, Link: https://parts.igem.org/Part:BBa_K124002), CCW12, PGK1 (BBa_K122000, Link:https://parts.igem.org/Part:BBa_K122000), HHF2, TEF1 controlling the ptxD_opt gene in S. cerevisiae. All previously described experiments were also performed using M2 medium with phosphite as phosphorus source.

T--Duesseldorf--Result3.Fig3klein.png

Figure 3: Growth of five S. cerevisiae strains containing ptxD_opt under the control of different promoters over 52 h in M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite. Strains with promoter TDH3 (blue), CCW12 (red), PGK1 (green), HHF2 (gray), TEF1 (yellow), measured at OD600.

All constructs with the five promoters show a constant, but slightly different growth in M2 medium containing phosphite (Figure 3). With the strongest promoter TDH3, S. cerevisiae achieves an OD600 of nearly 0.025, which is more than 100% more growth than the weakest promoter of the five: CCW12. The strains with the promoters PGK1 or TEF1 reach an OD600 of a little bit over 0.015, representing medium strength.


The final question is if our modified S. cerevisiae strains show a better growth on phosphite than the progenitor S. cerevisiae. Therefore, the growth of the strain with the ptxD_opt gene and the strongest promoter TDH3 was compared with the progenitor strain.

T--Duesseldorf--Result3.Fig4standartabweichungklein.png

Figure 4: Growth of the S. cerevisiae control strain (yellow) and S. cerevisiae with the ptxD_opt and TDH3 (blue) on M2 medium with 1.5% glucose, 0.5% ammonium sulfate, histidine (1.56 mg/l), leucine (380 mg/l) and lysine (1.52 mg/l) with phosphite over 52 h, measured at 600 nm. For the S. cerevisiae BY4742 background strain, uracil (18 mg/l) was added.

In Figure 4, the graph shows an initial growth of the progenitor strain S. cerevisiae, but the growth stops at an OD600 of less than 0.03 after 10 hours. After 10 hours, the cell density of S. cerevisiae starts to decrease, slowly in the beginning, but faster towards the end (around 50 h). At the end of the experiment the progenitor strain has an OD600 of less than 0.02. The modified S. cerevisiae strain with ptxD_opt under the control of the TDH3 promoter starts at nearly the same OD600 as the progenitor strain but after a short lag phase of around 5 hours, the strain grows, slowly in the beginning and faster towards the end, where it reaches an OD600 of over 0.02. Both strains start at the same OD600 level. The non-modified strain grows faster than the modified strain at the beginning, but then decreases more and more, while the modified strain needs some time but shows a slow increase of growth afterwards.


Conclusion

Stable dependencies between organisms are often based on nutrient exchange. Phosphorus is a macro nutrient essential for microorganisms. Creating a dependency based on the ability of S. cerevisiae to utilize an otherwise unusable phosphorus source like phosphite by expressing ptxD and making it metabolically available for E. coli is therefore a very promising approach.
In order to build a stable dependency, the other organisms in this system have to lack the enzymes for the catalysis from phosphite to phosphorus. The experiment with E. coli (Figure 1) and S. cerevisiae (Figure 2) demonstrates that neither E. coli nor S. cerevisiae show any growth in M2 medium with phosphite. Figure 3 shows the growth of engineered S. cerevisiae with different promoter_ptxD_opt constructs. All show growth regardless of what promoter was used but the promoter TDH3 showed the strongest growth rates. This indicates that our approach can work.
When comparing the growth of both S. cerevisiae strains (Figure 4), some differences can be detected. The non-modified S. cerevisiae grows much faster in the beginning but soon thereafter begins to die. The modified strain however shows slow but constant growth. As a consequence, we assume that the modified strain would outcompete contaminating microorganisms and possibly the other members of the co-culture. To add to that, we have designed our co-culture with more than one dependency, for example with nitrogen or carbon.
In general the growth was minimal, so for further experiments, higher concentrations of nutrients and phosphite might lead to better results. The slow growth rate of the modified strain is as we expected, because it is comparable to what the literature shows. There, a ptxD construct with the TEF1 promoter in S. cerevisiae was used and growth was monitored over 40 hours4. A constant temperature of 30°C also might increase growth. Moreover, a longer measurement time would also show the behavior of the culture over a longer time. This would be interesting because we would like to create a stable culture which can be maintained for as long as possible. In addition, co-culture experiments would likely lead to other results than monocultures. In this case it would be interesting to perform them as well with the same experimental design. For a future application in the co-culture, we suggest to use the phosphate exporter XPR1 from Homo sapiens5,6. It may help to provide for the other organisms, due to the export of the produced phosphate.


References

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https://www.sciencedirect.com/science/article/pii/S2211124713002684

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https://www.nature.com/articles/ng.3289