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| <h1 class="title">Storytelling:</h1> | | <h1 class="title">Storytelling:</h1> |
| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
− | We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX | + | We entered this project as the first Marburg iGEM team working with <i>Synechococcus elongatus</i> UTEX |
| 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us | | 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us |
| in front of many problems and questions. Especially the usage of different media, light conditions and other | | in front of many problems and questions. Especially the usage of different media, light conditions and other |
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| </p> | | </p> |
| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
− | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to | + | While we wanted to establish <i>Synechococcus elongatus</i> as a new chassis for the iGEM community and scientists we wanted to |
| show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore | | show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore |
| we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in | | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in |
− | light measurement, evaluated different reporters???, established a measurement method and compared it to a | + | light measurement, evaluated different reporters, established a measurement method and compared it to a |
− | already known FACS measurement method (?). | + | already known FACS measurement method. |
| </p> | | </p> |
| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
− | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. | + | 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 [link IHP] to confront this problem and standardize it. In the following | + | cyano and light <a href="https://2019.igem.org/Team:Marburg/Human_Practices">measurement experts</a> to confront this problem and standardize it. In the following |
| popup we show different ways of measurement, their (dis-)advantages and different results depending on the | | popup we show different ways of measurement, their (dis-)advantages and different results depending on the |
| measuring instrument.<br> | | measuring instrument.<br> |
| Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. | | Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. |
− | In literature and while talking with different experts (IHP), we recognized that small deviations of these | + | In literature and while talking with different experts, we recognized that small deviations of these |
| parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as | | parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as |
| a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters | | a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters |
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| Another aspect was measuring the expression and characterize our part. Different possibilities were | | Another aspect was measuring the expression and characterize our part. Different possibilities were |
| discussed and after testing them we decided on two methods in our project (plate reader and FACs). One | | discussed and after testing them we decided on two methods in our project (plate reader and FACs). One |
− | approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate | + | approach was to measure the fluorescence/luminescence with a plate reader. Plate |
| readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br> | | readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br> |
− | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In | + | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting). In |
| contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its | | contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its |
− | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not | + | own. On the other side not |
− | every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database | + | every laboratory posses a FACS/device. So in the end we would like to offer a two method analyzed database |
| from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the | | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the |
| difference in measurement methods.<br> | | difference in measurement methods.<br> |
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| We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology. What we often time stumbled upon when we were looking for literature on our iGEM project was that the information on light intensity in these papers were often inconsistent. Oftentimes the only values on the intensity were given in the unit µEinstein, but the needed details on how that number was measured, was missing. So some people would measure the intensities with a planar device, others would determine them via a spherical quantum sensor. | | We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology. What we often time stumbled upon when we were looking for literature on our iGEM project was that the information on light intensity in these papers were often inconsistent. Oftentimes the only values on the intensity were given in the unit µEinstein, but the needed details on how that number was measured, was missing. So some people would measure the intensities with a planar device, others would determine them via a spherical quantum sensor. |
| | | |
− | During our skype call with James Golden he emphasized that a lot of experiments are simply not reproducible, because there is no way to tell how much light one has to expose their organisms to. Additionally we got the feedback of Dr. Nicolas Schmelling that even professional cultivation devices from companies which are specialized on building them, can not deliver consistent and even illumination. | + | During our skype call with James Golden he emphasized that a lot of experiments are simply not reproducible, because there is no way to tell how much light one has to expose their organisms to. Additionally, we got the feedback of Dr. Nicolas Schmelling that even professional cultivation devices from companies which are specialized on building them, can not deliver consistent and even illumination. |
| To go even further, we think that the spectrum of the respective lamp should also be considered when talking about standardization. The light spectrum of our two lamps look as follows. | | To go even further, we think that the spectrum of the respective lamp should also be considered when talking about standardization. The light spectrum of our two lamps look as follows. |
− | [evtl. nur Bild der Spektren] | + | |
| Even though the standardization of the light quality seems to be a very hard task it should still be included in scientific works in order to give as much information as possible about the experimental setup. | | Even though the standardization of the light quality seems to be a very hard task it should still be included in scientific works in order to give as much information as possible about the experimental setup. |
− | We measured an equidistant grid of points at which we measured the average amount of photons (10 seconds) to minimize fluctuation. These data points were then interpolated with the help of a b spline surface to predict the amount of µmol photons at any given point of the incubator. This method is described in more detail on our model page.[Link zur Model page] We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology and immensely helps to create reproducible experimental setups. | + | We measured an equidistant grid of points at which we measured the average amount of photons (10 seconds) to minimize fluctuation. These data points were then interpolated with the help of a b spline surface to predict the amount of µmol photons at any given point of the incubator. This method is described in more detail on our <a href="https://2019.igem.org/Team:Marburg/Model">model</a> page. We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology and immensely helps to create reproducible experimental setups. |
− | We could show that light intensity had a big effect on reporter gene expression (FACS link) | + | We could show that light intensity had a big effect on reporter gene expression. |
| This displays the importance of standardization especially if one want to characterize parts such as promoters RBS terminator or engineer even more complex designs like genetic circuits or synthetic metabolic pathways. | | This displays the importance of standardization especially if one want to characterize parts such as promoters RBS terminator or engineer even more complex designs like genetic circuits or synthetic metabolic pathways. |
| We propose a standardization of the light measurement process and inclusion of information, such as the way of measuring, light source and proper light intensities in every publication for phototrophic organisms. | | We propose a standardization of the light measurement process and inclusion of information, such as the way of measuring, light source and proper light intensities in every publication for phototrophic organisms. |
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| <img src="https://static.igem.org/mediawiki/2019/6/65/T--Marburg--GrowthCurveOD.png" alt="GrowthCurveOD"> | | <img src="https://static.igem.org/mediawiki/2019/6/65/T--Marburg--GrowthCurveOD.png" alt="GrowthCurveOD"> |
| <figcaption> | | <figcaption> |
− | Fig.2 - Growth of S.elongatus UTEX 2973 and PCC 7942 measured by optical density. | + | Fig.2 - Growth of <i>S. elongatus</i> UTEX 2973 and PCC 7942 measured by optical density. |
| </figcaption> | | </figcaption> |
| </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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| 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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| <br> | | <br> |
| <br> | | <br> |
| + | </p> |
| + | <p> |
| <center><figure Style="text-align:center"> | | <center><figure Style="text-align:center"> |
| <!-- add this for centered pics--> | | <!-- add this for centered pics--> |
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| </figcaption> | | </figcaption> |
| </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 as shown in fig. XX to cultivate and measure |
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| 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 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. |
| | | |
− | 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 (read here what else we learned from <a href="https://2019.igem.org/Team:Marburg/Human_Practices">him</a>) 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. |
| | | |
| </p> | | </p> |