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| At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. | | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. |
| So we had to measure the light conditions in our incubators and while doing this simple task the first | | So we had to measure the light conditions in our incubators and while doing this simple task the first |
− | part of standardization began. We discovered that nearly every paper? is using different methods to measure | + | part of standardization began. We discovered that nearly every paper is using different methods to measure |
| their light conditions and that it is a really complex and important procedure. So we got in contact with | | their light conditions and that it is a really complex and important procedure. So we got in contact with |
| cyano and light measurement experts to confront this problem and standardize it. In the following | | cyano and light measurement experts to confront this problem and standardize it. In the following |
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| <p> | | <p> |
| Comparing flow cytometry measurements to optical density measurements we were able to find some striking differences.<br> | | Comparing flow cytometry measurements to optical density measurements we were able to find some striking differences.<br> |
− | Using the exact same probes and paying very close attention to work carefully we created to growth curves which, although showing the same tendency, differ from one another. While in the optical density measurements the culture seems to shift towards the stationary phase [Fig 2: Growth of S.elongatus UTEX 2973 and PCC 7942 measured by optical density], the cell counts show us a still exponentially growing culture [Fig 3: Growth of S.elongatus UTEX 2973 and PCC 7942 measured by cell count]. <br>Calculating the doubling time between two exact same time points for both approaches we were again able to find a difference: while the OD730 measurements resulted in a calculated doubling time of 108 minutes for the UTEX 2973 strain, the calculation using cell counts resulted in a doubling time of 94 minutes - a difference of 14 minutes between two measurement methods for the exact same samples! | + | Using the exact same probes and paying very close attention to work carefully we created to growth curves which, although showing the same tendency, differ from one another. While in the optical density measurements the culture seems to shift towards the stationary phase (Fig. 2), the cell counts show us a still exponentially growing culture (Fig. 3). <br>Calculating the doubling time between two exact same time points for both approaches we were again able to find a difference: while the OD730 measurements resulted in a calculated doubling time of 108 minutes for the UTEX 2973 strain, the calculation using cell counts resulted in a doubling time of 94 minutes - a difference of 14 minutes between two measurement methods for the exact same samples! |
| </p> | | </p> |
| <figure> | | <figure> |
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| <img src="https://static.igem.org/mediawiki/2019/9/99/T--Marburg--GrowthCurveCellCount.png" alt="CellCountSetup"> | | <img src="https://static.igem.org/mediawiki/2019/9/99/T--Marburg--GrowthCurveCellCount.png" alt="CellCountSetup"> |
| <figcaption> | | <figcaption> |
− | Fig.3 - Growth of S.elongatus UTEX 2973 and PCC 7942 measured by flow cytometry. | + | Fig.3 - Growth of <i>S. elongatus</i> UTEX 2973 and PCC 7942 measured by flow cytometry. |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <p> | | <p> |
| In our project we chose to use flow cytometry as an accurate method, to analyse gene expression levels of genetic constructs. <br> | | In our project we chose to use flow cytometry as an accurate method, to analyse gene expression levels of genetic constructs. <br> |
− | In an extensive experiment we assessed the fluorescence of a transformed YFP-construct in our cured strain, showing that the shuttle vector with the minimal replication element can be maintained in S. elongatus UTEX 2973.<br> | + | In an extensive experiment we assessed the fluorescence of a transformed YFP-construct in our cured strain, showing that the shuttle vector with the minimal replication element can be maintained in <i>S. elongatus</i> UTEX 2973.<br> |
| Using a similar setup as in our growth curve experiments, we analysed the strength of the fluorescence signal over time: <br><br> | | Using a similar setup as in our growth curve experiments, we analysed the strength of the fluorescence signal over time: <br><br> |
| As expected, no YFP expressing cells could be counted in the wild type strain. | | As expected, no YFP expressing cells could be counted in the wild type strain. |
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| For our project it was indispensable to establish a measurement workflow that is not only applicable | | For our project it was indispensable to establish a measurement workflow that is not only applicable |
| to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg | | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg |
− | Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement | + | Collection 2.0 with 55 parts we came to the conclusion it is also necessary to develop a measurement |
| method that suites such a large collection. Therefore we elaborated different workflows - containing | | method that suites such a large collection. Therefore we elaborated different workflows - containing |
| different cultivation vessels and parameters - and revised them after evaluating the results. In the end | | different cultivation vessels and parameters - and revised them after evaluating the results. In the end |
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| For the cultivating workflow we tested different well plate formats and growing parameters for the | | For the cultivating workflow we tested different well plate formats and growing parameters for the |
| best growing conditions. It was logistically the best way to cultivate and measure the parts in | | best growing conditions. It was logistically the best way to cultivate and measure the parts in |
− | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space | + | well plates, because the Marburg Collection 2.0 comprises 55 parts and we were limited in space |
| in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus | | in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus |
| elongatus</i> UTEX 2973 under our conditions since the cultures showed small | | elongatus</i> UTEX 2973 under our conditions since the cultures showed small |
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| because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. | | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. |
| Further it was necessary to use transparent wells to ensure every well with similar ight | | Further it was necessary to use transparent wells to ensure every well with similar ight |
− | conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) | + | conditions. Concerning of light conditions, we evaluated that the cells showed good |
| growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an | | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an |
| important role in cultivation of well plates cause the realtive small volumes and high surfaces | | important role in cultivation of well plates cause the realtive small volumes and high surfaces |
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| different seals for the well plates and in the end we came to the conclusion that using a | | different seals for the well plates and in the end we came to the conclusion that using a |
| semipermeable foil is the best solution. The evaporation could be minimalized and the cells were | | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were |
− | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to | + | able to get enough CO2 because air transfer was provided. By using a foil it was possible to |
| cultivate the cells for 2-3 days without losing significant amounts of medium. | | cultivate the cells for 2-3 days without losing significant amounts of medium. |
| <br> | | <br> |
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| </figure></center> | | </figure></center> |
| <br> | | <br> |
− | As described before we used the following workflow as shown in fig. XX to cultivate and measure | + | As described before we used the following workflow to cultivate and measure |
| our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the | | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the |
| end of the triparental conjugation. For every part we picked 3 different colonies and | | end of the triparental conjugation. For every part we picked 3 different colonies and |
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| A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was | | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was |
| inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while | | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while |
− | evaluating the results (that will be used as a blank while ...). When all the cultures in the | + | evaluating the results. When all the cultures in the |
| second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same | | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same |
| well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating | | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating |
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| “Strength and growth come only through continuous effort and struggle.” - Napoleon Hill | | “Strength and growth come only through continuous effort and struggle.” - Napoleon Hill |
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− | Although this quote was certainly never meant in this way, it is quite fitting to our project, as the growth of our Synechococcus elongatus strain UTEX 2973 was one of the key aspects throughout the year. | + | Although this quote was certainly never meant in this way, it is quite fitting to our project, as the growth of our <i>Synechococcus elongatus</i> strain UTEX 2973 was one of the key aspects throughout the year. |
| Our goal to create the fastest phototrophic chassis was fueled by our unwavered dream of accelerated research on the multitude of mechanisms and possibilities that phototrophic organisms have to offer. We were quick to learn that this goal was not as close as we might have thought. On our way we encountered countless obstacles, some easier to overcome than others - one of the most resilient ones being the growth conditions we had to provide. | | Our goal to create the fastest phototrophic chassis was fueled by our unwavered dream of accelerated research on the multitude of mechanisms and possibilities that phototrophic organisms have to offer. We were quick to learn that this goal was not as close as we might have thought. On our way we encountered countless obstacles, some easier to overcome than others - one of the most resilient ones being the growth conditions we had to provide. |
| Actually reaching the technical values we wanted was not the main issue, no, the hardest part was finding the holy grail of growth conditions, the perfect combination of parameters to cultivate our strain in. | | Actually reaching the technical values we wanted was not the main issue, no, the hardest part was finding the holy grail of growth conditions, the perfect combination of parameters to cultivate our strain in. |
| | | |
− | Digging through literature we found various different setups that were seemingly the “optimal growth conditions” for S.elongatus UTEX 2973 and it was apparent that in order to find the optimal conditions, we ultimately had to try all of them out by ourselves. So we set one of our biggest projects in motion, recording numerous different growth curves with many different parameters. | + | Digging through literature we found various different setups that were seemingly the “optimal growth conditions” for <i>S. elongatus</i> UTEX 2973 and it was apparent that in order to find the optimal conditions, we ultimately had to try all of them out by ourselves. So we set one of our biggest projects in motion, recording numerous different growth curves with many different parameters. |
| Before calibrating key parameters like CO2 concentration, light intensity and temperature, we conducted some smaller trials on various other criteria, such as lid type, flask size, flask type and culture volume, as those are not heavily affected by the other parameters. | | Before calibrating key parameters like CO2 concentration, light intensity and temperature, we conducted some smaller trials on various other criteria, such as lid type, flask size, flask type and culture volume, as those are not heavily affected by the other parameters. |
| Through these experiments, we could clearly identify a set that enabled the best growth for our chassis: plastic lids on 250ml erlenmeyer flasks with three chicanes and 50ml culture volume. Having fixed these initial parameters we set sail to the sea of endlessly variable growth conditions in hope to discover the true needs of S.elongatus UTEX 2973. | | Through these experiments, we could clearly identify a set that enabled the best growth for our chassis: plastic lids on 250ml erlenmeyer flasks with three chicanes and 50ml culture volume. Having fixed these initial parameters we set sail to the sea of endlessly variable growth conditions in hope to discover the true needs of S.elongatus UTEX 2973. |
− | As phototrophic chassis primarily require light and CO2 for their growth, those were the two parameters we were most interested in, but due to the UTEX 2973 strain being reportedly tolerant to higher temperatures than most other S.elongatus strains (Tan et al., 2018), this was another aspect to be tested. As time was scarce, we parallelized our measurements, meaning that while different temperatures or CO2 concentrations were put on trial we were able to compare the growth under different light intensities. | + | As phototrophic chassis primarily require light and CO2 for their growth, those were the two parameters we were most interested in, but due to the UTEX 2973 strain being reportedly tolerant to higher temperatures than most other <i>S. elongatus</i> strains (Tan et al., 2018), this was another aspect to be tested. As time was scarce, we parallelized our measurements, meaning that while different temperatures or CO2 concentrations were put on trial we were able to compare the growth under different light intensities. |
| | | |
− | At this point it is important to mention that the light intensities in our incubator were not always set the same way: in the beginning we measured the light distribution with a planar light measurement device, using a conversion chart we acquired from Prof. Dr. Annegret Wilde from Freiburg to convert the values to theoretical spherical values, but after our insightful talk to Prof. Dr. James W. Golden (read here what else we learned from him[link to Golden Skype Call]) we hurried to get hold of a spherical measurement device to make sure we could accurately set the light intensities - and the difference was striking: the doubling time of our cultures increased by a huge amount which was an important step into the right direction for us. | + | At this point it is important to mention that the light intensities in our incubator were not always set the same way: in the beginning we measured the light distribution with a planar light measurement device, using a conversion chart we acquired from Prof. Dr. Annegret Wilde from Freiburg to convert the values to theoretical spherical values, but after our insightful talk to Prof. Dr. James W. Golden (we hurried to get hold of a spherical measurement device to make sure we could accurately set the light intensities - and the difference was striking: the doubling time of our cultures increased by a huge amount which was an important step into the right direction for us. |
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| </p> | | </p> |