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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>S. 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 measurement experts 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 <i>Synechococcus elongatus</i>. 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 |
| that lead to the fastest growth speed.<br> | | that lead to the fastest growth speed.<br> |
| 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> |
− | At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 | + | At the end of the project we were able to create a protocol how to handle <i>Synechococcus elongatus</i> UTEX 2973 |
| and make a contribution to the cyano community by establishing essential/fixed standards in measurement. | | and make a contribution to the cyano community by establishing essential/fixed standards in measurement. |
| </p> | | </p> |
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| 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 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 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. |