Mguillaume (Talk | contribs) |
Mguillaume (Talk | contribs) |
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− | <img src="https:// | + | <img src="https://static.igem.org/mediawiki/2017/8/86/TAmsterdam_pm_logo.png"/> |
<a class="navbar-brand nav-link" href="https://2017.igem.org/Team:Amsterdam"> | <a class="navbar-brand nav-link" href="https://2017.igem.org/Team:Amsterdam"> | ||
Photosynthetic Magic | Photosynthetic Magic | ||
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<div class="summary-container"> | <div class="summary-container"> | ||
<div class="summary-col-left"> | <div class="summary-col-left"> | ||
− | <img class="animated-module-icon" src="https:// | + | <img class="animated-module-icon" src="https://static.igem.org/mediawiki/2017/6/63/TAmsterdam_shunt_icon.png"/> |
<br/> | <br/> | ||
</div> | </div> | ||
<div class="summary-col-mid"> | <div class="summary-col-mid"> | ||
<p class="summary-text"> | <p class="summary-text"> | ||
− | + | A major requisite of cyano-cell factories, according to expert's opinion, is that they must be able to produce in a stable fashion under industrial conditions. A recent quantitative analysis of the various ways to convert the energy of photons to chemical bonds has revealed that the direct utilization of sunlight is the most efficient [1]. This however means that cells will be exposed to diurnal regimes in which they will inevitably be exposed to periods of darkness. Our goal here is to achieve the first photoautotrophic cell factories that are able to stably produce fumarate around the clock. | |
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<br/> | <br/> | ||
</p> | </p> | ||
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</h1> | </h1> | ||
<p> | <p> | ||
− | < | + | <i> |
− | + | Synechocystis | |
− | + | </i> | |
− | + | does not naturally produce fumarate. However, model guided engineering found that removing a single gene within | |
− | + | <i> | |
− | + | Synechocystis | |
− | + | </i> | |
− | + | leads to a stable cell factory that produces fumarate as it grows during the day. Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome this challenge, we have taken a systems biology approach which interweaves theory, modeling, and experimentation to implement stable nighttime production of fumarate. We theorized that we can redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout of the | |
− | + | <i> | |
− | + | zwf | |
− | + | </i> | |
− | + | gene. Additionally, we also took inspiration from nature and speculated that the incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our models corroborate these predictions, however, they also suggest that the stability of the glyoxylate shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions. | |
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</p> | </p> | ||
</div> | </div> | ||
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2 | 2 | ||
</sub> | </sub> | ||
− | around the clock | + | around the clock Qp |
+ | <sub> | ||
+ | night | ||
+ | </sub> | ||
+ | of 12.7 µM grDW | ||
<sup> | <sup> | ||
-1 | -1 | ||
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-1 | -1 | ||
</sup> | </sup> | ||
− | + | Qp | |
+ | <sub> | ||
+ | day | ||
+ | </sub> | ||
+ | of 52.0 µM grDW | ||
<sup> | <sup> | ||
-1 | -1 | ||
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-1 | -1 | ||
</sup> | </sup> | ||
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</li> | </li> | ||
</ul> | </ul> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | Our cyano- | + | Our cyano-cell factory must be able to stably grow and produce under industrial conditions. At the industrial scale, |
<i> | <i> | ||
Synechocystis | Synechocystis | ||
</i> | </i> | ||
− | and other cyanobacteria are often grown in large outdoor ponds or in greenhouses [2], where natural solar radiation is the primary source of light. This means that the cultures are subject to an oscillating light-dark cycle. Therefore, we aim to make a cyanobacterial cell factory that is able to produce fumarate in a stable fashion during ,not only the day, but also the night. | + | and other cyanobacteria are often grown in large outdoor ponds or in greenhouses [2], where natural solar radiation is the primary source of light. This means that the cultures are subject to an oscillating light-dark cycle. Therefore, we aim to make a cyanobacterial cell factory that is able to produce fumarate in a stable fashion during, not only the day, but also the night. |
<br/> | <br/> | ||
We mimicked industrial conditions in the lab by tailoring commercially available photobioreactors (MC1000-OD, PSI, Czech Republic) to be capable of simulating dynamic white light regimes. This involved developing new algorithms to incorporate the on-line measurements (e.g. OD | We mimicked industrial conditions in the lab by tailoring commercially available photobioreactors (MC1000-OD, PSI, Czech Republic) to be capable of simulating dynamic white light regimes. This involved developing new algorithms to incorporate the on-line measurements (e.g. OD | ||
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"real-world" | "real-world" | ||
</a> | </a> | ||
− | beyond the academic | + | beyond the academic laboratory. |
</p> | </p> | ||
<p class="collapsible-main-header" id="stable"> | <p class="collapsible-main-header" id="stable"> | ||
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</p> | </p> | ||
<figure id="fig21"> | <figure id="fig21"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/9/9d/TAmsterdam_amsterdam_production_2.1.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</p> | </p> | ||
<figure id="fig22"> | <figure id="fig22"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/a/a8/TAmsterdam_amsterdam_production_2.2.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</b> | </b> | ||
<i> | <i> | ||
− | Cell growth and extracellular fumarate production in different | + | Cell growth and extracellular fumarate production in different Synechocystis strains. (a) Cell growth of both wild type and ΔfumC in Multi-Cultivator under constant light illumination for over 200 hours. (b) Extracellular fumarate production for both strains. Error bars indicate the standard deviations. |
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</i> | </i> | ||
</figcaption> | </figcaption> | ||
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</i> | </i> | ||
strain seems to hold up to scrutiny. | strain seems to hold up to scrutiny. | ||
− | + | </p> | |
+ | <p> | ||
We also calculated the carbon partitioning towards fumarate in the | We also calculated the carbon partitioning towards fumarate in the | ||
<i> | <i> | ||
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</p> | </p> | ||
<figure id="fig23"> | <figure id="fig23"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/a/a0/TAmsterdam_amsterdam_production_2.3.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</b> | </b> | ||
<i> | <i> | ||
− | Strict relationship between growth and product formation in the | + | Strict relationship between growth and product formation in the Δ fumC strain. (a) A linear relationship between growth rate and biomass specific fumarate productivity. Each point represents a single observation, and solid line is a linear fit of all experimental data points. Dash line is based on in silico FBA simulations of the genome-scale metabolic model of Synechocystis using biomass maximization as the objective function. (b) Carbon partitioning of fumarate production on biomass at different light regimes. Error bars indicate the standard deviation of carbon partitioning calculated at times throughout the cultivation (n>3). |
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</i> | </i> | ||
</figcaption> | </figcaption> | ||
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</p> | </p> | ||
<figure id="fig24"> | <figure id="fig24"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/1/15/TAmsterdam_amsterdam_production_2.4.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</i> | </i> | ||
does not grow without sunlight, the growth coupled production strategy of fumarate is not possible during the night. Thus, we expect no growth coupled fumarate production. But is there another strategy in which we can exploit the night to produce fumarate? | does not grow without sunlight, the growth coupled production strategy of fumarate is not possible during the night. Thus, we expect no growth coupled fumarate production. But is there another strategy in which we can exploit the night to produce fumarate? | ||
− | + | </p> | |
+ | <p> | ||
In order to survive the night, | In order to survive the night, | ||
<i> | <i> | ||
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</i> | </i> | ||
produces energy by catabolizing its glycogen storage [24]. The TCA cycle is often associated with this process as it provides the electron carriers for ATP production via respiration. As fumarate is an intermediate metabolite in the TCA cycle, increasing the carbon flux through the TCA cycle could be exploited to increase fumarate production during the night. | produces energy by catabolizing its glycogen storage [24]. The TCA cycle is often associated with this process as it provides the electron carriers for ATP production via respiration. As fumarate is an intermediate metabolite in the TCA cycle, increasing the carbon flux through the TCA cycle could be exploited to increase fumarate production during the night. | ||
− | + | </p> | |
+ | <p> | ||
An unexpected challenge, probably quite unique to photoautotrophs, that emerged is that flux measurements experimentally determined that | An unexpected challenge, probably quite unique to photoautotrophs, that emerged is that flux measurements experimentally determined that | ||
<i> | <i> | ||
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</sub> | </sub> | ||
which was so costly to fix in the first place! | which was so costly to fix in the first place! | ||
− | + | </p> | |
+ | <p> | ||
To increase the flux through the TCA cycle, we took a modeling approach, which predicted that we could increase flux towards the TCA cycle by deletion of the | To increase the flux through the TCA cycle, we took a modeling approach, which predicted that we could increase flux towards the TCA cycle by deletion of the | ||
<i> | <i> | ||
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batch experiment | batch experiment | ||
</a> | </a> | ||
− | and compared the production capacity of these | + | and compared the production capacity of these strain, during the day and night phases. We earlier |
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<a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Produce#methods1" target="_blank"> | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Produce#methods1" target="_blank"> | ||
showed | showed | ||
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</p> | </p> | ||
<figure id="fig25"> | <figure id="fig25"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/8/8c/TAmsterdam_amsterdam_production_2.5.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</b> | </b> | ||
<i> | <i> | ||
− | PCR confirmation of gene deletions on Wild Type and | + | PCR confirmation of gene deletions on Wild Type and Δ fumC background of Δ zwf and Δ fumC Δ zwf. A clean knockout gives a single DNA band at 2202 bp, while for wild type (WT) this is 3.430 bp. As a positive control (C+) the knockout plasmid was used and as negative control (C-) the mazF plasmid was used. Most importantly the Wild Type (WT) gene is not present anymore in our strains, which implies that the strains are fully segregated knock outs. |
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</i> | </i> | ||
</figcaption> | </figcaption> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | From the diurnal batch culture, we were able to calculate the daytime production for the 4 strains. After | + | From the diurnal batch culture, we were able to calculate the daytime production for the two strains (N=4 for both strains). After 72 hours at an OD |
<sub> | <sub> | ||
720 | 720 | ||
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day | day | ||
</sub> | </sub> | ||
− | of 58. | + | of 58.7 µM grDW |
<sup> | <sup> | ||
-1 | -1 | ||
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fumC | fumC | ||
</i> | </i> | ||
− | and | + | and 52.0 µM grDW |
<sup> | <sup> | ||
-1 | -1 | ||
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</p> | </p> | ||
<figure id="fig26"> | <figure id="fig26"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/c/c0/TAmsterdam_amsterdam_production_2.6.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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day | day | ||
</sub> | </sub> | ||
− | of the different strains. | + | of the different strains. After 72h. This experiment has been carried out with similar results 4 times independently for |
<i> | <i> | ||
Δ | Δ | ||
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zwf | zwf | ||
</i> | </i> | ||
− | and | + | and 5 times for the |
<i> | <i> | ||
Δ | Δ | ||
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fumC | fumC | ||
</i> | </i> | ||
− | strain. We can see that already | + | strain. We can see that already after 64 hours, the |
<i> | <i> | ||
Δ | Δ | ||
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night | night | ||
</sub> | </sub> | ||
− | of | + | of 12.5 µM grDW |
<sup> | <sup> | ||
-1 | -1 | ||
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night | night | ||
</sub> | </sub> | ||
− | of | + | of 5.2 µM grDW |
<sup> | <sup> | ||
-1 | -1 | ||
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</p> | </p> | ||
<p> | <p> | ||
− | It must be noted however, that nighttime fumarate production can be perceived as finite since it relies on the glycogen storages that were built up during the day. The reported QP is calculated by looking at the night as whole. Although the comparisons between the strains are still valid, it is important to understand that QP is not necessarily constant throughout the night as it depends on the dynamics of glycogen catabolism.The | + | It must be noted however, that nighttime fumarate production can be perceived as finite since it relies on the glycogen storages that were built up during the day. The reported QP is calculated by looking at the night as whole. Although the comparisons between the strains are still valid, it is important to understand that QP is not necessarily constant throughout the night as it depends on the dynamics of glycogen catabolism. |
+ | </p> | ||
+ | <p> | ||
+ | The | ||
<i> | <i> | ||
Δ | Δ | ||
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</p> | </p> | ||
<figure id="fig27"> | <figure id="fig27"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/7/7c/TAmsterdam_amsterdam_production_2.7.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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night | night | ||
</sub> | </sub> | ||
− | of the | + | of the two strains after 64h. This experiment has been carried out with similar results 4 times independently for |
<i> | <i> | ||
Δ | Δ | ||
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zwf | zwf | ||
</i> | </i> | ||
− | and | + | and 5 times for the |
<i> | <i> | ||
Δ | Δ | ||
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</figure> | </figure> | ||
<figure id="fig28"> | <figure id="fig28"> | ||
− | <img class="module-figure-image" src="https:// | + | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/d/dc/TAmsterdam_amsterdam_production_2.8.png"/> |
<figcaption class="module-figure-text"> | <figcaption class="module-figure-text"> | ||
<b> | <b> | ||
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</b> | </b> | ||
<i> | <i> | ||
− | Fumarate production of the four different strains during the night. The fumarate production value is not corrected by OD. This confirms that the | + | Fumarate production of the four different strains during the night. The fumarate production value is not corrected by OD. This confirms that the ΔfumCΔzwf and the ΔfumC strain produce fumarate at night. |
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p class="project-header"> | ||
+ | Overall production in conditions mimicking industrial settings | ||
+ | </p> | ||
+ | <p> | ||
+ | The productivity of our strains in an industrial setting is the combined production of day and night. We calculated the Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | over the course of a 24h period (figure 2.9). We find that the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | has a Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | of 26.6 µM grDW | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | hour | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | , while the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | has a Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | of 23.8 µM grDW | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | hour | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | . We can thus conclude that the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | produces more fumarate over the course of a natural day. This clearly shows the benefit of having a day/night production system (and it is extremely gratifying to see that all our modeling and experimental efforts were not in vain!) | ||
+ | </p> | ||
+ | <figure id="fig29"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/3/3b/TAmsterdam_amsterdam_production_2.9.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.9 | ||
+ | </b> | ||
+ | <i> | ||
+ | Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | of the two different strains after 72 hours. This experiment has been carried out with similar results 4 times independently for ΔfumCΔzwf and 5 times for the ΔfumC. ΔfumCΔzwf has a higher Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | than the ΔfumC. WT and Δzwf do not produce fumarate. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure id="table21"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/f/f0/TAmsterdam_amsterdam_production_table21.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Table 2.1 | ||
+ | </b> | ||
+ | <i> | ||
+ | Fumarate production parameters for four different strains. Qp values are in µM grDW | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | hour | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | measured after 72 hours. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p class="collapsible-main-header" id="conc2"> | ||
+ | Conclusion | ||
+ | </p> | ||
+ | <p> | ||
+ | We showed that the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | and the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | are both able to produce fumarate during the daytime using the growth coupled strategy. During the night, the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | produces more fumarate than the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | , which confirms the extensive | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Model#ppp" target="_blank"> | ||
+ | modeling | ||
+ | </a> | ||
+ | we did for this part of the project. We can confirm that at night the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | is forced to direct carbon from glycogen catabolism towards the TCA cycle to form fumarate. We thus engineered a | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | cell factory that is able to produce fumarate around the clock, using two different production strategies - both stable - one for day and another for night. On top of this, since we used only knock-outs and did not resort to the cloning of heterologous genes, the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | will be a stable production strain for many generations to come. As a bonus, the higher nighttime production of the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | compared to the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | does imply that by knocking out the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | , we force flux to the TCA cycle. This is an important finding, as it opens up opportunities for the nighttime production of valuable TCA cycle intermediates in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | . To our knowledge such a diurnal, dual strategy, photoautotrophic cell factory has never been reported before. | ||
+ | </p> | ||
+ | <p class="collapsible-main-header" id="methods2"> | ||
+ | Methods | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Strain construction: | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | and | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | and segregation | ||
+ | </p> | ||
+ | <p> | ||
+ | The | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | gene encodes glucose-6-phosphate 1-dehydrogenase, which catalyses the first step in the Pentose Phosphate Pathway. We knocked out the | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | gene in the Wild Type and the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | background, to construct the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | and the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | mutants. We used the | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Methods" target="_blank"> | ||
+ | Markerless knock out method | ||
+ | </a> | ||
+ | . The homologous regions of the | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | gene were amplified from the | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | genomic DNA, with Herculase polymerase using primers BP1, BP2, BP3 and BP4. The | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Methods" target="_blank"> | ||
+ | biobrick T vector | ||
+ | </a> | ||
+ | used was the pFL-AN. Resulting in plasmid in | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | knockout plasmids, which were used for the first and second round of transformation | ||
+ | </p> | ||
+ | <p class="project-header" id="char-zwf"> | ||
+ | Characterising | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | , | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | and | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | </p> | ||
+ | <p> | ||
+ | In order to characterise the different | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | strains, we performed different cultivation experiments. We simultaneously performed a batch and a turbidostat experiment in a modified Multi-Cultivator under a photonfluxostat light regime, as described in Du et al. 2016[4] and outlined on our methods page. Our | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | strains were cultivated in BG-11 medium which contained 10 mM TES KOH buffer. For the batch experiment, we had 4 vessels that contained | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | and 4 vessels that contained | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | . In the turbidostat set up, we cultivated four strains i) Wild Type, ii) | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | , iii) | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | , and iv) | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | all in duplicates. The light intensity per OD followed a sinusoidal regime to simulate day/night cycles yielding 16 hours of darkness (0 μE s | ||
+ | <sup> | ||
+ | -2 | ||
+ | </sup> | ||
+ | OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | ) and 8 hours of light (peaking at 120 μE s | ||
+ | <sup> | ||
+ | -2 | ||
+ | </sup> | ||
+ | OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | ), calculated by equation 2.1, where t is the time in hours. | ||
+ | </p> | ||
+ | <p> | ||
+ | \[2.1 \frac{\mu E}{s^{2}}="240\sin" (2\pi\cdot (\frac{t}{24}+\frac{1}{4}) )-120\] | ||
+ | </p> | ||
+ | <p> | ||
+ | This equation returns negative values during the period, so they are clamped at a minimum value of 0. All cultures are inoculated at an initial OD | ||
+ | <sub> | ||
+ | 720 | ||
+ | </sub> | ||
+ | of 0.05 and were grown at a constant light intensity of 20 μE until all vessels reached an OD | ||
+ | <sub> | ||
+ | 720 | ||
+ | </sub> | ||
+ | of 0.6. At this point, we switched the light output to the designated light regime. | ||
+ | </p> | ||
+ | <p> | ||
+ | Instead of the more commonly adopted 12h day/12h night, we chose a 8h day/ 16h night as indicated above. While longer days would have probably allowed us to reach higher levels of production in the lab, after visiting an actual | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/HP/Gold_Integrated" target="_blank"> | ||
+ | production facility | ||
+ | </a> | ||
+ | , we were convinced that this would not be representative of a real-world scenario. The structures surrounding the greenhouses in many production plans provide shading during dawn and dusk. This makes the sun rise somewhat later, and set somewhat sooner, for production photoautotrophs. Our light regime in the lab mimics this, and is yet another factor that confers credibility to the actual production numbers that we report.. | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Sampling and fumarate measurements | ||
+ | </p> | ||
+ | <p> | ||
+ | During the course of the experiment, we took samples at every perceived dawn and dusk. After sampling we had to determine the concentration of fumarate. 1 ml of sample was centrifuged at 15.000 rpm for 10 min. Then 500 μl supernatant was taken and filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.22 μm) for sample preparation. Fumarate concentration was measured by HPLC-UV/VIS (LC-20AT, Prominence, Shimadzu), with ion exclusion Rezex ROA-Organic Acid column (250x4.6 mm; Phenomenex) and UV detector (SPD-20A, Prominence, Shimadzu) at 210 nm wavelength. 50 μL of the HPLC samples were injected through an autosampler (SIL-20AC, Prominence, Shimadzu), with 5 mM H | ||
+ | <sub> | ||
+ | 2 | ||
+ | </sub> | ||
+ | SO | ||
+ | <sub> | ||
+ | 4 | ||
+ | </sub> | ||
+ | as eluent at a flow rate of 0.15 ml min | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | and column temperature of 45 ℃. Fumarate retention time was determined as 18.16 and 18.36 min and fumarate samples were normalised by a correction factor composed of 10 mM divided by the measured TES concentration. | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Production calculations | ||
+ | </p> | ||
+ | <p> | ||
+ | To calculate the fumarate production during the day and the fumarate production during the night. We made the following assumptions: i) | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | does not grow in the absence of sunlight, so does not grow during the night. ii) The production of fumarate during the day is growth coupled. We calculated the fumarate yield during the day, yield | ||
+ | <sub> | ||
+ | day | ||
+ | </sub> | ||
+ | by | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | fumarate/ | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | OD in mmol OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | over the course of 1 day in the batch culture. By dividing this number by 8 hours, we could calculate Qp | ||
+ | <sub> | ||
+ | day | ||
+ | </sub> | ||
+ | in mmol OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | h | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | for the day. During the night, no cell growth was assumed, therefore we expected no change in OD, however as | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | physiology changes at night, this can influence the scattering of the light and thereby the OD measurement can change during the night. To account for this effect, we determined the night time fumarate yield, yield | ||
+ | <sub> | ||
+ | night | ||
+ | </sub> | ||
+ | as | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | fumarate/mean OD. By dividing this number by 16 hours we could calculate Qp | ||
+ | <sub> | ||
+ | night | ||
+ | </sub> | ||
+ | in mmol OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | h | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | . The overall 24h fumarate production could be determined by knitting together the nighttime production and the daytime production. we determined the yield | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | as yield | ||
+ | <sub> | ||
+ | day | ||
+ | </sub> | ||
+ | plus yield | ||
+ | <sub> | ||
+ | night | ||
+ | </sub> | ||
+ | . Dividing this number by 24 hours, we could determine Qp | ||
+ | <sub> | ||
+ | daily | ||
+ | </sub> | ||
+ | in mmol OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | hour | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | . To transform these QPs to a more familiar unit, we multiplied all QP's by a conversion factor that converts OD | ||
+ | <sub> | ||
+ | 720 | ||
+ | </sub> | ||
+ | to gram dry weight (148 mg L | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | OD | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | [16] ) . We then receive fumarate QPs in mM gDW | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | hour | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | . | ||
+ | </p> | ||
+ | <p> | ||
+ | 2.2 \[yield_{day} ="\frac{\triangle" [fumarate]}{\triangle OD}\] 2.3 \[yield_{night} ="\frac{\triangle" [fumarate]}{mean OD}\] 2.4 \[Qp_{day} ="\frac{yield_{day}}{8" \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1}\] 2.5 \[Qp_{night}="\frac{yield_{night}}{16" \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1} \] 2.6 \[Qp_{daily} ="\frac{yield_{day}+yield_{night}}{24" \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1}\] | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="module-collapsible-container-outer" id="fourth"> | ||
+ | <p class="in-text-link" id="fourth" onclick="displayCollapsible(this.id)"> | ||
+ | ▶ Beyond the native metabolic network of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | </p> | ||
+ | <div class="module-collapsible-container-inner" id="fourth" style="display:none;"> | ||
+ | <p class="collapsible-main-header" id="intro3"> | ||
+ | Introduction | ||
+ | </p> | ||
+ | <p> | ||
+ | Being able to force the cell to direct carbon flux towards the reactions of the native TCA cycle in the | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | zwf | ||
+ | </i> | ||
+ | strain increases the fumarate production during the night. However, by knocking out fumarase, we disrupted the cyclic nature of the TCA, which may no longer operate as a cycle. This construct yields one fumarate per glycogen catabolized. We started wondering if there could be a way to use synthetic biology to improve the nighttime production efficiency in terms of carbon usage. We turned to nature for inspiration… | ||
+ | <br> | ||
+ | Many microorganisms express a glyoxylate shunt. The glyoxylate shunt consists of two enzymes which are not natively present in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | : isocitrate lyase (ICL) and malate synthase (MS). The first enzyme, ICL, catalyzes the reaction of isocitrate into glyoxylate and succinate, where succinate can then be consumed by succinate dehydrogenase to produce fumarate and FADPH2. As for glyoxylate, this compound is then consumed by MS, along with acetyl-CoA, to make malate. Malate is then converted to oxaloacetate by malate dehydrogenase, yielding NADPH (fig. 2.11). | ||
+ | </br> | ||
+ | </p> | ||
+ | <figure id="fig211"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/8/83/TAmsterdam_amsterdam_production_2.11.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.11 | ||
+ | </b> | ||
+ | <i> | ||
+ | The glyoxylate shunt with | ||
<i> | <i> | ||
− | + | ΔfumC | |
</i> | </i> | ||
+ | . The glyoxylate shunt is comprised of two enzymes: isocitrate lyase (ICL) and malate synthase (MS). The orange X signifies the knockout of the | ||
<i> | <i> | ||
fumC | fumC | ||
</i> | </i> | ||
+ | gene. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | We hypothesized that if a synthetic glyoxylate shunt was introduced into | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | , this would not only reconnect the stunted | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | TCA cycle, but also increase the flux towards fumarate production, by feeding into reactions which produce electron carriers - one of the main roles of the TCA cycle [17]. This would potentially align fumarate production with an increase in fitness during the night, providing the type of positive selection pressure, which could be used to stabilize the expression of this heterologous pathway in a production strain as advised by | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/HP/Gold_Integrated" target="_blank"> | ||
+ | experts | ||
+ | </a> | ||
+ | . | ||
+ | </p> | ||
+ | <p> | ||
+ | We have explored this idea by introducing the glyoxylate shunt into the genome-scale metabolic model that had been guiding our metabolic engineering strategies thus far. We found that our hypothesis is corroborated by | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Model" target="_blank"> | ||
+ | model simulations | ||
+ | </a> | ||
+ | . However, timing the activation of this pathway turned out to be of the essence when engineering a stable strain. This led us to the construction of the first fully-segregated (i.e all copies of the chromosome have the same allele) promoter library in a polyploid organism such as the cyanobacterium we work with ( | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | ), but not without first developing a method to do so. | ||
+ | </p> | ||
+ | <p class="collapsible-main-header" id="results3"> | ||
+ | Results and discussion | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Modeling the shunt | ||
+ | </p> | ||
+ | <p> | ||
+ | We explored the possibilities for the introduction of the glyoxylate shunt in terms of fitness-gain and fumarate production, by running simulations in which the glyoxylate shunt had been "cloned" into our ur metabolic engineering strategies thus far. We found that our hypothesis is corroborated by | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Model" target="_blank"> | ||
+ | model | ||
+ | </a> | ||
+ | (Oh! If only it was that easy in the lab as well…) | ||
+ | </p> | ||
+ | <p> | ||
+ | We found that the glyoxylate shunt must be well timed: it cannot be active during the day, as the glyoxylate shunt will draw carbon from more prefered pathways. We are thus faced with another challenge: how do we time the expression of the glyoxylate shunt such that the cell has a fitness advantage, while partitioning more carbon to fumarate synthesis? Clearly, only expressing the glyoxylate shunt during the night with the right expression levels is hard. The expression level and timing of a gene is, at the very least, partially regulated through its promoter. That is why we aim to look for a promoter with the right expression pattern for the shunt genes to be activate only in the night. Photoreceptor-based transcriptional circuits could at a first glance be suitable control systems. However, the delays between transcriptional activation (or repression) and the actual increase (or drop) in enzyme levels can be many hours depending for instance on protein stability. Such delay could be sufficient to remove the competitive edge of harboring the genes encoding the shunt, and therefore, rendering it phenotypically unstable. | ||
+ | <br> | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | harbors circadian clocks with periods close to 24 h [18]. Chances are, that there already is a promoter present in its chromosome with the right expression levels at the right time for our glyoxylate shunt enzymes. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. The rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes, hinting at the presence of an appropriate promoter for our shunt genes. To express the glyoxylate shunt only during the night and with the right expression levels, we aim to use what nature has to offer and make a glyoxylate shunt promoter library. | ||
+ | </br> | ||
+ | </p> | ||
+ | <figure id="fig212"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/e/e3/TAmsterdam_amsterdam_production_2.12.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.12 | ||
+ | </b> | ||
+ | <i> | ||
+ | The promoter library DNA is prepared by cutting up the genome of | ||
<i> | <i> | ||
− | + | Synechocystis | |
</i> | </i> | ||
+ | into fragments that have on average the size of a promoter (100-1000bp). Each of these fragments will be introduced upstream of the glyoxylate shunt genes, resulting in thousands of unique plasmids. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p class="project-header"> | ||
+ | Making the library | ||
+ | </p> | ||
+ | <p> | ||
+ | The glyoxylate shunt promoter library was constructed by cutting up the genome of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | into fragments that have, on average, the size of a promoter (between 100-1000 bp). Subsequently, each of these fragments was introduced upstream of the glyoxylate shunt genes (fig. 2.12) and transformed to | ||
+ | <i> | ||
+ | E. coli | ||
+ | </i> | ||
+ | . A colony PCR with primers flanking the promoter region (seg_h2_fw and ms_col_rv) confirmed that all inserts had a different length (fig. 2.13). Once we were happy with our odds (99.9%) that every possible base pair is in the library at least once, the DNA was extracted from | ||
+ | <i> | ||
+ | E. coli | ||
+ | </i> | ||
+ | . We required our final library to be integrated into the the genome at the neutral site | ||
+ | <i> | ||
+ | slr0168 | ||
+ | </i> | ||
+ | of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | to ensure that the difference in observed phenotype can be attributed to the difference in promoter, rather than something else (e.g. changes in plasmid copy number). However, the introduction of the promoter library into | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | was easier said than done. | ||
+ | </p> | ||
+ | <figure id="fig213"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/e/e2/TAmsterdam_amsterdam_production_2.13.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.13 | ||
+ | </b> | ||
+ | <i> | ||
+ | After transforming the library DNA into | ||
<i> | <i> | ||
− | + | E. coli | |
</i> | </i> | ||
− | and the | + | , a colony PCR with primers flanking the promoter region confirmed that every insert had a different length. |
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | is a polyploid, each cell containing 4 to 20 copies of the genome depending on the physiological state and environmental conditions [19]. Polyploidy poses a challenge in creating a stable mutant strain, since the newly introduced genes have to be "fully segregated" (i.e. present in all chromosome copies) to avoid that they revert to wild type in future generations. However, a fully segregated promoter library in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | is something that has not yet been reported and poses a major challenge - How does one guarantee that all members of the library are fully segregated? - Our supervisor, told us that we had three options: | ||
+ | </p> | ||
+ | <ul class="produce-list"> | ||
+ | <li> | ||
+ | Quit here; | ||
+ | </li> | ||
+ | <li> | ||
+ | Colony PCR tens of thousands of colonies; | ||
+ | </li> | ||
+ | <li> | ||
+ | Or come up with a new method to do it. | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p> | ||
+ | We chose the latter and decided to develop a method to create a fully segregated (promoter) libraries in polyploid organisms. | ||
+ | <br> | ||
+ | The strategy we developed employs the | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Method" target="_blank"> | ||
+ | markerless method | ||
+ | </a> | ||
+ | that facilitates the selection for fully segregated colonies. This procedure is based on the negative selection method using the toxic gene | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | [20]. The | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | gene is an endoribonuclease that is regulated by a nickel-induced element (NIE) [21], killing the cells upon exposure to elevated concentrations of nickel. The | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | cassette needs to be introduced into the neutral site | ||
+ | <i> | ||
+ | slr0168 | ||
+ | </i> | ||
+ | of the | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | genome, enabling the introduction of the glyoxylate shunt into the neutral site at later time points, replacing the | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | cassette itself. With this selection method, complete segregation of the glyoxylate shunt in the genome can be tested simply by adding nickel - if the colony is not fully segregated, even if there is only one copy of the | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | -cassette in the genome, the cell will be killed upon exposure to nickel! However, when a colony is fully segregated, and all the | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | cassettes are replaced by glyoxylate shunt genes, the colony remains unharmed. Furthermore, we wanted the glyoxylate shunt promoter library to represent the entire genome (i.e. as many colonies as possible to reach a probability of having all the positions in the chromosome in the library at least once approaching 100%). To expand the library, we allowed the cells time to segregate before exposing them to nickel, as cells often shift towards full segregation over time [22]. A positive selection marker was used to push cells towards full segregation. However, high amounts of antibiotics might hamper cell growth and thereby slow down segregation. Studying the segregation dynamics, we found the sweet-spot between antibiotic concentration and the timing before exposing cells to nickel: after transformation (day 0), cells were supplemented with kanamycin (50 μg/ml) on day 1 and nickel 20 μM on day 4 (fig. 2.14). | ||
+ | <br> | ||
+ | Using our newly developed method, we created the first fully segregated library representing the entire genome (99.9% confidence) of | ||
<i> | <i> | ||
− | + | Synechocystis | |
</i> | </i> | ||
+ | upstream of the glyoxylate shunt genes. What is more; the library is now ready to be tested. The beauty of the glyoxylate shunt promoter library approach lies in the fact that expressing the shunt at both the appropriate time and level increases fitness. Therefore, we can now easily select the ideal promoter using long term cultivations and letting Darwinian selection do the rest. The promoter that expresses the shunt the most optimally will be the one that grows the fastest, outcompeting the rest of the population. This will also be the best fumarate producer, which will eventually be used to further increase the nighttime fumarate production. | ||
+ | </br> | ||
+ | </br> | ||
+ | </p> | ||
+ | <figure id="fig214"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/d/de/TAmsterdam_amsterdam_production_2.14.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.14 | ||
+ | </b> | ||
+ | <i> | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p class="collapsible-main-header" id="conc3"> | ||
+ | Conclusion | ||
+ | </p> | ||
+ | <p> | ||
+ | Using the genome-scale metabolic model, we found that the glyoxylate shunt can increase the growth rate of our fumarate producing strain, but only under specific conditions: (i) it cannot be active during the day, as this will result in a decrease in fitness, and (ii) the expression of glyoxylate shunt enzymes need to be at the right levels. In order to find a promoter with the right expression characteristics for the glyoxylate shunt, we developed a method to create a fully segregated promoter library in polyploid organisms, such as our own cyanobacterium | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | . Using our own developed method, we created the first fully segregated library representing the entire genome (99.9% confidence) of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | upstream of the glyoxylate shunt genes. This library is now ready to be tested to further increase nighttime fumarate production. | ||
+ | </p> | ||
+ | <p class="collapsible-main-header" id="methods3"> | ||
+ | Methods | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Construction of the glyoxylate shunt genes in a plasmid | ||
+ | </p> | ||
+ | <p> | ||
+ | The glyoxylate shunt genes are derived from | ||
+ | <i> | ||
+ | Chlorogloeopsis fritschii | ||
+ | </i> | ||
+ | PCC 9212 and are obtained from the Bryant Lab in a plasmid called pAQ1Ex_cpc_MS-ICL_Sp [23]. To get both genes expressed under the same promoter, the glyoxylate shunt will be placed in a plasmid (pFLXN-h1h2-rbs-yfp-0168; obtained from MMP) that contains a ribosomal binding site (rbs) in front of each gene and a BglII restriction site where the genomic DNA fragments will be inserted. To push segregation and to select in | ||
+ | <i> | ||
+ | E. coli | ||
+ | </i> | ||
+ | , a kanamycin (Kan) resistance cassette was introduced in the plasmid. | ||
+ | </p> | ||
+ | <p> | ||
+ | Both plasmids were digested with NcoI and BamHI (Thermo Scientific FastDigest), placed on a 1% agarose gel, cut out and purified (Qiagen PCR/gel purification kit). Subsequently, the insert (MS-ICL) was ligated in the vector (pFLXN-h1h2-rbs), replacing the YFP fragment using T4 ligase and transformed to DH5alpha competent cells. Colonies were confirmed with colony PCR using primers h2_down_rv and h1_up_fw. The confirmed colonies were inoculated in LB (+50 μg/mL Kan), after which the plasmid, now called pFLXN-h1h2-rbs-MS-ICL (fig. 2.15), was extracted and sequence confirmed by Macrogen Europe (the Netherlands). | ||
+ | </p> | ||
+ | <figure id="fig215"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/c/cd/TAmsterdam_amsterdam_production_2.15.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.15 | ||
+ | </b> | ||
+ | <i> | ||
+ | Plasmid map of pFLXN-H1H2-rbs-MS-rbs-ICL containing the glyoxylate shunt genes with homologous regions to be integrated into the neutral site | ||
<i> | <i> | ||
− | + | slr0168 | |
</i> | </i> | ||
− | + | . Genomic DNA fragments were inserted at the BglII site. | |
</i> | </i> | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
+ | <p class="project-header"> | ||
+ | Method to create a promoter library in polyploid organisms | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | <i> | ||
+ | Construction of the glyoxylate shunt promoter library DNA using E. coli | ||
+ | </i> | ||
+ | </p> | ||
+ | <p> | ||
+ | The construction of a promoter library consists of multiple steps. First, the genomic DNA of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | was | ||
+ | <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Methods" target="_blank"> | ||
+ | extracted | ||
+ | </a> | ||
+ | on a large scale (20 mL, OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | ~5) and purified using RNAse. Then, the purified genomic DNA was digested with Sau3AI (Thermo Scientific FastDigest), yielding fragments ranging from 100-1000 bp. These fragments will be referred to as the inserts. | ||
+ | <br> | ||
+ | To open pFLXN-h1h2-rbs-MS-ICL, enabling it to integrate the inserts, the plasmid was digested with BglII which is compatible with Sau3AI (Thermo Scientific FastDigest; incubated for 3 hours, added Phosphatase after 1.5 hour to prevent self ligation). | ||
+ | <br> | ||
+ | Next, the insert was ligated in the vector using a ligation ratio of 1:7 (v:i) and transformed into Dh5-Alpha Competent E. coli cells (MCLAB) multiple times, yielding it total over 100,000 positive colonies. This led to a probability of 99.9% that any fragment in the genome will occur at least once in the library. | ||
+ | </br> | ||
+ | </br> | ||
+ | </p> | ||
+ | <figure id="box21"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/9/99/TAmsterdam_amsterdam_production_box21.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Box 2.1 | ||
+ | </b> | ||
+ | <i> | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | The colonies were washed of the plates, stored as glycerol stocks (15% glycerol; -80℃) and inoculated overnight. The plasmids were extracted using a MiniPrep (Qiagen) and stored as the promoter library DNA. | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | <i> | ||
+ | Segregation Dynamics in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | </i> | ||
+ | </p> | ||
+ | <p> | ||
+ | In order to obtain a fully segregated library that represents the entire genome of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | , the segregation dynamics was studied, aiming to find the sweet-spot between kanamycin concentration and timing before exposing the library to Nickel. | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | Transformation | ||
+ | </p> | ||
+ | <p> | ||
+ | A pre-culture (fully segregated | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | - | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | Ω) of 100 mL (20 mL per transformation) was used (OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | = 1). The culture was spun down (3900 rpm; 10 min), washed twice with fresh BG-11 to remove possible antibiotics, and concentrated 100 times to a volume of 1 mL (200 μL per transformation) (OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | = 100). Next, the library DNA was added (30 μg DNA/mL culture) to 800 μL culture. As a negative control, 200 μL of culture without DNA was used. The cultures were incubated for 5 hours (30 ℃; ~30 μmol/m2/s of constant white light), after which the 200 μL per transformed culture was diluted in fresh BG-11(20 mL) without antibiotics and incubated (~22 hours )(day 0). Subsequently, kanamycin was added in the following concentrations to push segregation (day 1): | ||
+ | </p> | ||
+ | <ul class="produce-list"> | ||
+ | <li> | ||
+ | negative control: 40 μg/mL Kan | ||
+ | </li> | ||
+ | <li> | ||
+ | Kan40: 40 μg/mL Kan | ||
+ | </li> | ||
+ | <li> | ||
+ | Kan50: 50 μg/mL Kan | ||
+ | </li> | ||
+ | <li> | ||
+ | Kan75: 75 μg/mL Kan | ||
+ | </li> | ||
+ | <li> | ||
+ | Kan100: 100 μg/mL Kan | ||
+ | </li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <p class="project-header"> | ||
+ | <i> | ||
+ | Droplet experiment | ||
+ | </i> | ||
+ | </p> | ||
+ | <figure id="table22"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/7/74/TAmsterdam_amsterdam_production_table22.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Table 2.2 | ||
+ | </b> | ||
+ | <i> | ||
+ | The ability of clones to grow (x) on different BG-11 plates. Concentrations: Kanamycin: 50 μg/mL; Nickel: 20 mM. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | Every day, the cells were plated on the three different BG-11 plates using a dilution series droplet design (fig. 2.16). | ||
+ | <br> | ||
+ | The OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | of the cultures were measured, after which the dilution series was prepared using a 96 well-plate and a multichannel pipet. First, all the BG-11 was added in appropriate amounts to the wells following the pipetting scheme, after which the inoculum from the different cultures was added to row A. After mixing properly, the dilution series were made by pipetting 10 μL from row A to B. After mixing, 10 μL was pipetted from row B to C, and so on, resulting in a dilution series that contained two rows per culture: | ||
+ | <br/> | ||
+ | <br/> | ||
+ | 10 | ||
+ | <sup> | ||
+ | 0 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -1 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -2 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -3 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -4 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -6 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -7 | ||
+ | </sup> | ||
+ | . | ||
+ | <br/> | ||
+ | 10 | ||
+ | <sup> | ||
+ | -0.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -1.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -2.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -3.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -4.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -5.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -6.5 | ||
+ | </sup> | ||
+ | , 10 | ||
+ | <sup> | ||
+ | -7.5 | ||
+ | </sup> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | From each well, 5 μL will be plated in droplets on the three types of BG-11 plates. | ||
+ | </br> | ||
+ | </p> | ||
+ | <figure id="fig216"> | ||
+ | <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/4/43/TAmsterdam_amsterdam_production_2.16.png"/> | ||
+ | <figcaption class="module-figure-text"> | ||
+ | <b> | ||
+ | Figure 2.16 | ||
+ | </b> | ||
+ | <i> | ||
+ | Pipetting scheme in order to make the droplet dilution series and the plating design. | ||
+ | </i> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <p> | ||
+ | Transformation efficiency can be calculated by dividing the amount of colonies on the BG-11 plate by the amount of colonies on the BG-11 + Kan plate. The ratio of fully segregated colonies can be calculated by dividing the amount of colonies on the BG-11 + Kan plate by the amount of colonies on the BG-11 + Kan + Nickel plate. If all clones are fully segregated, this will result in a ratio of 1. Segregation efficiency can be calculated by dividing the amount of colonies on the BG-11 plate by the amount of colonies on the BG-11 + Kan + Nickel plate. | ||
+ | </p> | ||
+ | <p class="project-header"> | ||
+ | <i> | ||
+ | Large scale transformation of the glyoxylate shunt promoter library DNA into | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | </i> | ||
+ | </p> | ||
+ | <p> | ||
+ | From the segregation dynamics, the transformation and segregation efficiency was calculated to estimate the amount of transformations needed to represent the entire genome. A pre-culture ( | ||
+ | <i> | ||
+ | Δ | ||
+ | </i> | ||
+ | <i> | ||
+ | fumC | ||
+ | </i> | ||
+ | - | ||
+ | <i> | ||
+ | mazF | ||
+ | </i> | ||
+ | Ω) of 20 mL per transformation was used (OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | between 1 and 2). The culture was spun down (3900 rpm; 10 min), washed twice with fresh BG-11 to remove antibiotics, and concentrated 100 times to a volume of 200 μL per transformation. Next, the library DNA was added (30 μg DNA/mL culture) and incubated for 5 hours (30 ℃; ~30 μmol/m2/s of constant white light). After five hours, the 200 μL transformed culture was diluted in fresh BG-11(20 mL) without antibiotics and incubated for ~22 hours (day 0). Subsequently, Kanamycin was added (50 μg/mL) to push segregation (day 1) and cultivated for an additional three days. At day 4, the culture was spun down (3900 rpm; 10 min) and concentrated (OD | ||
+ | <sub> | ||
+ | 730 | ||
+ | </sub> | ||
+ | ~40), after which 100 μL per plate was spread on BG-11 plates (1% agar) containing Kanamycin (50 μg/mL) and Nickel (20 μM). The plates were incubated for 8 days (30 ℃; ~30 μmol/m2/s of constant white light) until colonies appeared, resulting in the first fully segregated glyoxylate shunt promoter library in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | . | ||
+ | </p> | ||
+ | </br> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="module-visible-container"> | ||
+ | <h1> | ||
+ | References | ||
+ | </h1> | ||
+ | <ol class="references-list"> | ||
+ | <li> | ||
+ | Lips, D., Schuurmans, J. M. M., dos Santos, F. B., & Hellingwerf, K. J. (2017). Many ways towards "solar fuel": Quantitative analysis of the most promising strategies and the main challenges during scale-up. Energy & Environmental Science. | ||
+ | </li> | ||
+ | <li> | ||
+ | René H. Wijffels, Olaf Kruse, and Klaas J. Hellingwerf. "Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae". In: Current Opinion in Biotechnology 24.3 (2013), pp. 405-413. | ||
+ | </li> | ||
+ | <li> | ||
+ | Patrik R. Jones. "Genetic Instability in Cyanobacteria - An Elephant in the Room?" In: Frontiers in Bioengineering and Biotechnology 2.May (2014), pp. 1-5. | ||
+ | </li> | ||
+ | <li> | ||
+ | Wei Du, S. Andreas Angermayr, Joeri A. Jongbloets, Douwe Molenaar, Herwig Bachmann, Klaas J. Hellingwerf, and Filipe Branco dos Santos. "Nonhierarchical flux regulation exposes the fitness burden associated with lactate production in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | sp. PCC6803". In: ACS Synthetic Biology (2016), acssynbio.6b00235. | ||
+ | </li> | ||
+ | <li> | ||
+ | Wei Du, Joeri A. Jongbloets, Coco van Boxtel, Hugo Pineda Hernandez, David Lips, Brett G. Oliver, Klaas J. Hellingwerf, and Filipe Branco dos Santos. "Alignment of microbial fitness with engineered product formation: Obligatory coupling between acetate production and photoautotrophic growth". 2017. | ||
+ | </li> | ||
+ | <li> | ||
+ | Teusink B, Smid EJ. Modelling strategies for the industrial exploitation of lactic acid bacteria. Nat Rev Microbiol. 2006;4:46-56 | ||
+ | </li> | ||
+ | <li> | ||
+ | Darmon E, Leach DR. Bacterial genome instability. Microbiol Mol Biol Rev. 2014;78:1-39. | ||
+ | </li> | ||
+ | <li> | ||
+ | Renda BA, Hammerling MJ, Barrick JE. Engineering reduced evolutionary potential for synthetic biology. Mol Biosyst. 2014;10:1668-78. | ||
+ | </li> | ||
+ | <li> | ||
+ | Feist AM, Zielinski DC, Orth JD, Schellenberger J, Herrgard MJ, Palsson BO. Model-driven evaluation of the production potential for growth-coupled products of | ||
+ | <i> | ||
+ | Escherichia coli | ||
+ | </i> | ||
+ | . Metab Eng. 2010;12:173-86 | ||
+ | </li> | ||
+ | <li> | ||
+ | Erdrich P, Knoop H, Steuer R, Klamt S. Cyanobacterial biofuels: new insights and strain design strategies revealed by computational modeling. Microb Cell Fact. 2014;13:128. | ||
+ | </li> | ||
+ | <li> | ||
+ | Nogales, J., Gudmundsson, S., Knight, E. M., Palsson, B. O., & Thiele, I. (2012). Detailing the optimality of photosynthesis in cyanobacteria through systems biology analysis. Proceedings of the National Academy of Sciences, 109(7), 2678-2683. | ||
+ | </li> | ||
+ | <li> | ||
+ | Bachmann H, Molenaar D, Branco dos Santos F, Teusink B. Experimental evolution and the adjustment of metabolic strategies in lactic acid bacteria. FEMS Microbiol Rev. 2017;41 Supp_1:S201-19. | ||
+ | </li> | ||
+ | <li> | ||
+ | Bryson V, Szybalski W. Microbial Selection. Science. 1952;116:45-51. | ||
+ | </li> | ||
+ | <li> | ||
+ | Angermayr, S. A. & Hellingwerf, K. J. On the Use of Metabolic Control Analysis in the Optimization of Cyanobacterial Biosolar Cell Factories. J. Phys. Chem. B (2013). doi:10.1021/jp4013152 | ||
+ | </li> | ||
+ | <li> | ||
+ | Ni Wan, Drew M. DeLorenzo, Lian He, Le You, Cheryl M. Immethun, George Wang, Ed- ward E.K. Baidoo, Whitney Hollinshead, Jay D. Keasling, Tae Seok Moon, and Yinjie J. Tang. "Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence". In: Biotechnology and Bioengineering 114.7 (2017), pp. 1593-1602. | ||
+ | </li> | ||
+ | <li> | ||
+ | Du, W., Jongbloets, J. A., Hernandez, H. P., Bruggeman, F. J., Hellingwerf, K. J., & dos Santos, F. B. (2016). Photonfluxostat: A method for light-limited batch cultivation of cyanobacteria at different, yet constant, growth rates. Algal Research, 20, 118-125. | ||
+ | </li> | ||
+ | <li> | ||
+ | Lee J. Sweetlove, Katherine F M Beard, Adriano Nunes-Nesi, Alisdair R. Fernie, and R. George Ratcliffe. "Not just a circle: Flux modes in the plant TCA cycle". In: Trends in Plant Science 15.8 (2010), pp. 462-470 | ||
+ | </li> | ||
+ | <li> | ||
+ | Tu, Benjamin P., and Steven L. McKnight. "Metabolic cycles as an underlying basis of biological oscillations." Nature reviews Molecular cell biology 7.9 (2006): 696-701. | ||
+ | </li> | ||
+ | <li> | ||
+ | Biology, C. & Soppa, J. Microbiology The ploidy level of | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | sp. PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters. (2016) | ||
+ | </li> | ||
+ | <li> | ||
+ | Cheah, Y.E., Albers, S.C. & Peebles, C.A.M. A novel counter-selection method for markerless genetic modification in | ||
+ | <i> | ||
+ | Synechocystis | ||
+ | </i> | ||
+ | sp. PCC 6803. Biotechnol. Prog. 29, 23-30 (2013). | ||
+ | </li> | ||
+ | <li> | ||
+ | Lopez-maury, L., Garcia-dominguez, M., Florencio, F. J. & Reyes, J.C. A two-component signal transduction system involved in nickel sensing in the cyanobacterium. 43, 247-256 (2002). | ||
+ | </li> | ||
+ | <li> | ||
+ | Griese, M. & Lange, C. Ploidy in cyanobacteria. 323, 124-131 (2011). | ||
+ | </li> | ||
+ | <li> | ||
+ | Zhang, Shuyi, and Donald A. Bryant. "Biochemical validation of the glyoxylate cycle in the cyanobacterium | ||
+ | <i> | ||
+ | Chlorogloeopsis fritschii | ||
+ | </i> | ||
+ | strain PCC 9212." Journal of Biological Chemistry 290.22 (2015): 14019-14030. | ||
+ | </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div class="module-collapsible-container-outer" id="last"> | ||
+ | <p class="in-text-link" id="last" onclick="displayCollapsible(this.id)"> | ||
+ | ▶ Supplementary Material | ||
+ | </p> | ||
+ | <div class="module-collapsible-container-inner" id="last" style="display:none;"> | ||
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+ | <img alt="Matrix" class="footer-sponsor-img" id="matrix" src="https://static.igem.org/mediawiki/2017/5/57/TAmsterdam_matrix.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="http://www.merckmillipore.com/" style="order:10" target="_blank"> | ||
+ | <img alt="Merck" class="footer-sponsor-img" id="merck" src="https://static.igem.org/mediawiki/2017/e/e5/TAmsterdam_merck.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="http://rivm.nl/en" style="order:14" target="_blank"> | ||
+ | <img alt="Rivm" class="footer-sponsor-img" id="rivm" src="https://static.igem.org/mediawiki/2017/f/f3/TAmsterdam_rivm.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="http://phenomenex.com/" style="order:10" target="_blank"> | ||
+ | <img alt="Phenomenex" class="footer-sponsor-img" id="phenomenex" src="https://static.igem.org/mediawiki/2017/0/02/TAmsterdam_phenomenex.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="http://www.snapgene.com/" style="order:6" target="_blank"> | ||
+ | <img alt="Snapgene" class="footer-sponsor-img" id="snapgene" src="https://static.igem.org/mediawiki/2017/8/80/TAmsterdam_snapgene.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="http://sils.uva.nl/" style="order:8" target="_blank"> | ||
+ | <img alt="Sils" class="footer-sponsor-img" id="sils" src="https://static.igem.org/mediawiki/2017/e/e4/TAmsterdam_sils.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="https://www.unitedconsumers.com/" style="order:7" target="_blank"> | ||
+ | <img alt="United consumers" class="footer-sponsor-img" id="unitedconsumers" src="https://static.igem.org/mediawiki/2017/2/2d/TAmsterdam_united_consumers.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="https://www.vu.nl/en/" style="order:2" target="_blank"> | ||
+ | <img alt="Vu" class="footer-sponsor-img" id="vu" src="https://static.igem.org/mediawiki/2017/5/5a/TAmsterdam_vu.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="https://science.vu.nl/en/index.aspx" style="order:4" target="_blank"> | ||
+ | <img alt="Vu fs" class="footer-sponsor-img" id="vufs" src="https://static.igem.org/mediawiki/2017/3/38/TAmsterdam_vu_fs.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | <a href="https://www.cs.vu.nl/en/" style="order:13" target="_blank"> | ||
+ | <img alt="Vu cs" class="footer-sponsor-img" id="vucs" src="https://static.igem.org/mediawiki/2017/c/ca/TAmsterdam_vu_cs.png"> | ||
+ | </img> | ||
+ | </a> | ||
+ | </div> | ||
+ | <!--// | ||
+ | <div class="footer-col-right"> | ||
+ | <a href="mailto:igem17.ams@gmail.com" target="_top"> | ||
+ | <img src="/images/email_logo.jpg" class="footer-sponsor-img"> | ||
+ | </a> | ||
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+ | <img src="/images/facebook_logo.png" class="footer-sponsor-img"> | ||
+ | </a> | ||
+ | </div> | ||
+ | //--> | ||
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</div> | </div> | ||
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</div> | </div> | ||
</html> | </html> | ||
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Latest revision as of 19:52, 15 December 2017
A major requisite of cyano-cell factories, according to expert's opinion, is that they must be able to produce in a stable fashion under industrial conditions. A recent quantitative analysis of the various ways to convert the energy of photons to chemical bonds has revealed that the direct utilization of sunlight is the most efficient [1]. This however means that cells will be exposed to diurnal regimes in which they will inevitably be exposed to periods of darkness. Our goal here is to achieve the first photoautotrophic cell factories that are able to stably produce fumarate around the clock.
Produce
Overview
Synechocystis does not naturally produce fumarate. However, model guided engineering found that removing a single gene within Synechocystis leads to a stable cell factory that produces fumarate as it grows during the day. Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome this challenge, we have taken a systems biology approach which interweaves theory, modeling, and experimentation to implement stable nighttime production of fumarate. We theorized that we can redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout of the zwf gene. Additionally, we also took inspiration from nature and speculated that the incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our models corroborate these predictions, however, they also suggest that the stability of the glyoxylate shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.
Highlights
- Engineered a Δ fumC Δ zwf Synechocystis strain, that uses different fumarate production strategies during day and night.
- Developed a method to make fully segregated libraries in polyploid organisms
- Created the first fully segregated library representing the entire genome (99.9% confidence) of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to further increase nighttime fumarate production.
- Stable production of fumarate directly from CO 2 around the clock Qp night of 12.7 µM grDW -1 hour -1 Qp day of 52.0 µM grDW -1 hour -1
▶ Industrial conditions
▶ Stable daytime production - Δ fumC
▶ Stable nighttime production - Δ fumC Δ zwf
▶ Beyond the native metabolic network of Synechocystis
References
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▶ Supplementary Material