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| <div class="popup-container"> | | <div class="popup-container"> |
| <div class="popup-header"> | | <div class="popup-header"> |
− | <h1 class="title">Reporter</h1> | + | <h1 class="title">Reporters</h1> |
| <button type="button" onclick="hide('reporter')">X</button> | | <button type="button" onclick="hide('reporter')">X</button> |
| </div> | | </div> |
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| When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable elements to characterize BioBricks. For a good characterization a suitable reporter is required. But reporters can be more than just merely a detection tool for transcriptional activity but they can also give a deeper insight into cellular conditions beyond the genetic context. We provide a <a href="https://2019.igem.org/Team:Marburg/Results#marburg_collection" target="_blank">diverse set of reporters</a> not only for the purpose of <a href="https://2019.igem.org/Team:Marburg/Improve">describing genetic tools</a> but also for the sensing of a variety of parameters which are crucial for cyanobacteria. | | When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable elements to characterize BioBricks. For a good characterization a suitable reporter is required. But reporters can be more than just merely a detection tool for transcriptional activity but they can also give a deeper insight into cellular conditions beyond the genetic context. We provide a <a href="https://2019.igem.org/Team:Marburg/Results#marburg_collection" target="_blank">diverse set of reporters</a> not only for the purpose of <a href="https://2019.igem.org/Team:Marburg/Improve">describing genetic tools</a> but also for the sensing of a variety of parameters which are crucial for cyanobacteria. |
| + | <br> <br> |
| + | As an addition to the fluorescent reporters, we included a set of luminescent reporters to bring measurements of genetic constructs to a new level as they mostly bypass autofluorescence from cyanobacterial cells. The red-shifted version of NanoLuc -namely teLuc- bears the potential of the best reporter in <i>S. elongatus</i> as its absorbance bypasses autofluorescent signals better than NanoLuc and shows a higher relative bioluminescent signal<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678970/" target="_blank">(Yeh <i>et al.</i>, 2017)</a>. |
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| </p> | | </p> |
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| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
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− | Furthermore, it is possible to determine the cell volume and size, as well as to distinguish between different kinds of cells, particles or cell clumps. For this, there are scatter detectors around the capilar. A forward scatter detector (FSC) and a stream side scatter detector (SSC) are placed around the stream.<br><br> | + | Furthermore, it is possible to determine the cell volume and size, as well as to distinguish between different kinds of cells, particles or cell clumps. For this, there are scatter detectors around the capillary. A forward scatter detector (FSC) and a stream side scatter detector (SSC) are placed around the stream.<br><br> |
| </p> | | </p> |
− | | + | <p> |
− | <h2 class="subtitle" style="font-size: 1.5rem !important; text-align:center">Flow cytrometry for growth curves</h2> | + | <u>Flow cytrometry for growth curves</u> |
| + | </p> |
| <p> | | <p> |
| With the flow cytometry device available to us we were able to capture highly accurate cell counts. This brought us the idea of implementing this technique in a way less related to fluorescent reporters: counting cells in our cultures to capture growth curves instead of relying on optical density measurements. <br><br> | | With the flow cytometry device available to us we were able to capture highly accurate cell counts. This brought us the idea of implementing this technique in a way less related to fluorescent reporters: counting cells in our cultures to capture growth curves instead of relying on optical density measurements. <br><br> |
| Measurements of optical density are highly influenced by a multitude of factors. When measuring samples it is common to receive different results every time the same sample is measured, as in the meantime the distribution of cells inside of the sample has changed - mainly due to them slowly sinking to the bottom of the cuvette while not being shaken. This leads to high measurement errors. <br><br> | | Measurements of optical density are highly influenced by a multitude of factors. When measuring samples it is common to receive different results every time the same sample is measured, as in the meantime the distribution of cells inside of the sample has changed - mainly due to them slowly sinking to the bottom of the cuvette while not being shaken. This leads to high measurement errors. <br><br> |
− | Cell counts offer a promising alternative, as they are independent of factors that could influence OD measurements: impurity in the sample can distort OD measurements, while in flow cytometry the polluting particles will mostly be clearly distinguishable from the other counts. Especially with cyanobacterial cultures this can be used as a huge advantage: the autofluorescence of the cell gives us a clear way to select for the right event counts in our device, which we can then gate in order to receive just the number of cells with the fitting fluorescence signal.<br><br> | + | Cell counting offers a promising alternative, as they are independent of factors that could influence OD measurements: Impurity in the sample can distort OD measurements, while in flow cytometry the polluting particles will mostly be clearly distinguishable from the other counts. Especially with cyanobacterial cultures this can be used as a huge advantage: the autofluorescence of the cell gives us a clear way to select for the right event counts in our device, which we can then gate in order to receive just the number of cells with the fitting fluorescence signal.<br><br> |
| In order to construct an actual growth curve out of this, another important part is needed: counting beads. The beads emit a distinct fluorescent signal that can be clearly detected and distinguished from other events. Using different filters we can select for the fluorescence we want to look at, i.e. the fluorescence of the beads or the fluorescence of our cyanobacteria. The event number of the counting beads can now be used to determine the exact number of cells in the culture - this is how it works: | | In order to construct an actual growth curve out of this, another important part is needed: counting beads. The beads emit a distinct fluorescent signal that can be clearly detected and distinguished from other events. Using different filters we can select for the fluorescence we want to look at, i.e. the fluorescence of the beads or the fluorescence of our cyanobacteria. The event number of the counting beads can now be used to determine the exact number of cells in the culture - this is how it works: |
| <br><br> | | <br><br> |
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| <img src="https://static.igem.org/mediawiki/2019/f/f2/T--Marburg--CellCountSetup.png" > | | <img src="https://static.igem.org/mediawiki/2019/f/f2/T--Marburg--CellCountSetup.png" > |
| <figcaption> | | <figcaption> |
− | Fig. 1 - Setup for the creation of growth curves through cell counts with flow cytometry. | + | Fig. 1 - Setup for the creation of growth curves through cell counting with flow cytometry. |
| </figcaption> | | </figcaption> |
| </figure></center> | | </figure></center> |
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| Comparing flow cytometry measurements to optical density measurements we were able to find some striking differences. | | Comparing flow cytometry measurements to optical density measurements we were able to find some striking differences. |
− | Using the exact same samples 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). 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 samples and paying very close attention to work carefully we created two 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). 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> |
| <center><figure Style="text-align:center"> | | <center><figure Style="text-align:center"> |
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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 <i>S. elongatus</i> 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.<br><br> |
| </figcaption> | | </figcaption> |
| </figure></center> | | </figure></center> |
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| <center><figure Style="text-align:center"> | | <center><figure Style="text-align:center"> |
| <!-- add this for centered pics--> | | <!-- add this for centered pics--> |
− | <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="CellCountFlowCytometry"> |
| <figcaption> | | <figcaption> |
| Fig.3 - Growth of <i>S. elongatus</i> 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. |
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| <br><br> | | <br><br> |
− | <h2 class="subtitle" style="font-size: 1.5rem !important; text-align:center" >Cell cytometry to examine gene expression levels</h2> | + | <p> |
| + | <u>Cell cytometry to examine gene expression levels</u> |
| + | </p> |
| <p> | | <p> |
− | In our project we chose to use flow cytometry as an accurate method, to analyse gene expression levels of genetic constructs. | + | In our project we chose to use flow cytometry as an accurate method, to analyze gene expression levels of genetic constructs. |
| 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. | | 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. |
− | Using a similar setup as in our growth curve experiments, we analysed the strength of the fluorescence signal over time: | + | Using a similar setup as in our growth curve experiments, we analyzed the strength of the fluorescence signal over time: |
| 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. |
| </p> | | </p> |
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| <br><br> | | <br><br> |
| <p> | | <p> |
− | For the conjugant strain it was obvious that a steady fluorescent signal could be obtained. For a lower light intensity the strength of the signal stayed the same throughout the whole experiment, while at higher light intensities a shift towards higher fluorescence intensities could be observed. | + | For the conjugant strain it was obvious that a steady fluorescent signal could be obtained. For a lower light intensity the strength of the signal stayed the same throughout the whole experiment, while at higher light intensities a wider range of fluorescence intensities could be observed. |
| </p> | | </p> |
| <br><br> | | <br><br> |
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| best growing conditions. Due to the size of the Marburg Collection 2.0 (55 parts) and space limitation | | best growing conditions. Due to the size of the Marburg Collection 2.0 (55 parts) and space limitation |
| in our incubator, our first thought was using well-plates. We started with 96-well-plates and found out that it was impossible to cultivate <i>Synechococcus | | in our incubator, our first thought was using well-plates. We started with 96-well-plates and found out that it was impossible to cultivate <i>Synechococcus |
− | elongatus</i> UTEX 2973 in incubator with 130 rpm. The rpm of | + | elongatus</i> UTEX 2973 in an incubator with 130 rpm. The rpm of |
| the incubator was limited because cultures in flasks had to be incubated at the same time, presenting risks of | | the incubator was limited because cultures in flasks had to be incubated at the same time, presenting risks of |
| falling over at higher rpm. After revising the workflow over and over we came to | | falling over at higher rpm. After revising the workflow over and over we came to |
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| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
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− | Additionally, it was necessary to use transparent wells to ensure every well with similar light | + | Additionally, it was necessary to use transparent wells to ensure every well would be provided with similar light |
| conditions. Concerning the light conditions, we evaluated that the cells showed good | | conditions. Concerning the light conditions, we evaluated that the cells showed good |
| growth in the wells at low-light conditions (around 500 µE). The evaporation of medium played an | | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium played an |
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| 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 thanks to improvement in air circulation. By using a foil it was possible to | | able to get enough CO2 thanks to improvement in air circulation. By using a foil it was possible to |
− | cultivate the cells for 2-3 days without losing significant amount of media. | + | cultivate the cells for 2-3 days without losing significant amounts of media. |
| <br> | | <br> |
| <br> | | <br> |
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| <p> | | <p> |
| <br> | | <br> |
− | As described before we used the following workflow as shown in Fig. 1 to cultivate and measure | + | As described before we used the workflow as shown in Fig. 1 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 transformed via triparental conjugation. For every part we picked three different colonies and |
− | end of the triparental conjugation. For every part we picked three different colonies and
| + | |
| inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates | | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
− | we could inoculate eight different parts with three biological parallels. When the cultures grew to | + | we could inoculate eight different parts with three biological replicates. When the cultures grew to |
| OD<sub>730</sub>=0.6-0.8 they were inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells | | OD<sub>730</sub>=0.6-0.8 they were inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells |
| 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 an OD<sub>730</sub>= 0.1 untransformed culture that was used as a blank while |
| evaluating the results. 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 |
− | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When | + | technical replicates of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When |
| the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred | | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
| into a 96-well-plate. Every well of the 24-well-plate was measured three | | into a 96-well-plate. Every well of the 24-well-plate was measured three |
− | times. Following this workflow we were able to measure three biological parallels and | + | times. Following this workflow we were able to measure three biological replicates and |
− | two technical parallels for every biological parallel. It enabled us to have a good statistical | + | two technical replicates for every biological replicate. It enabled us to have a good statistical |
| database and gives our results a stronger meaning and significance. While working with this workflow | | database and gives our results a stronger meaning and significance. While working with this workflow |
| it was essential to keep the cultures in their exponential phase because it would significantly | | it was essential to keep the cultures in their exponential phase because it would significantly |
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| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
| | | |
− | Concerning the measurement part we decided to transfer the cultures into black/white luminescence | + | For the measurement we decided to transfer the cultures into black/white well plates and luminescence |
− | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every | + | was measured in white ones. We measured in 96-well-plates because it enabled us to measure every |
| part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Furthermore we could measure | | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Furthermore we could measure |
| eight parts simultaneously in one plate. (four 24-well-plates lead into one 96-well-plate for | | eight parts simultaneously in one plate. (four 24-well-plates lead into one 96-well-plate for |
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| </p> | | </p> |
| <p> | | <p> |
− | <h2 class="subtitle" style="font-size: 1.5rem !important; text-align:center" text-align=center>Fluorescence measurement</h2> | + | <u>Fluorescence measurement</u> |
| + | </p> |
| <p> | | <p> |
| After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. | | After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. |
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| measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the | | measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the |
| border of the well showed consistent results with small standard deviations. We used the | | border of the well showed consistent results with small standard deviations. We used the |
− | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as | + | same settings for all fluorescence measurements. While using sYFP as a |
| signal for our part measurement we have set the emission wavelength to 515 nm and the excitation | | signal for our part measurement we have set the emission wavelength to 515 nm and the excitation |
− | wavelength to 527 nm, fitting the exact wavelengths of the sYFP.<br> | + | wavelength to 527 nm, fitting the exact wavelengths of sYFP.<br> |
| <br> | | <br> |
| </p> | | </p> |