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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 capilar. A forward scatter detector (FSC) and a stream side scatter detector (SSC) are placed around the stream.<br><br> |
| </p> | | </p> |
− | <h2>Flow cytrometry for growth curves</h2> | + | <h2 class="subtitle">Flow cytrometry for growth curves</h2> |
| <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> |
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| As one can already see from the formula, usually 50µl of beads are added to each sample that is run through the flow cytometer. This allows for accurate comparability.<br> | | As one can already see from the formula, usually 50µl of beads are added to each sample that is run through the flow cytometer. This allows for accurate comparability.<br> |
| Figure 1 shows our setup for the measurement of growth curves. The gated beads are counted to an event number of 1000. Meanwhile our cells are counted in a defined gate reaching from 2x10^3 to 10^5 relative fluorescence units. For detection of autofluorescence the APC filter was used. APC stands for Allophycocyanin, as this filter is designed to show the fluorescence of excited Allophycocyanin from red algae - a protein similar to phycocyanin in cyanobacteria, which is the reason why this setup works well to show cyanobacterial autofluorescence.<br><br> | | Figure 1 shows our setup for the measurement of growth curves. The gated beads are counted to an event number of 1000. Meanwhile our cells are counted in a defined gate reaching from 2x10^3 to 10^5 relative fluorescence units. For detection of autofluorescence the APC filter was used. APC stands for Allophycocyanin, as this filter is designed to show the fluorescence of excited Allophycocyanin from red algae - a protein similar to phycocyanin in cyanobacteria, which is the reason why this setup works well to show cyanobacterial autofluorescence.<br><br> |
− | | + | </p> |
| <figure> | | <figure> |
| <img src="https://static.igem.org/mediawiki/2019/f/f2/T--Marburg--CellCountSetup.png" alt="CellCountSetup"> | | <img src="https://static.igem.org/mediawiki/2019/f/f2/T--Marburg--CellCountSetup.png" alt="CellCountSetup"> |
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| </figure> | | </figure> |
| | | |
− | | + | <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: 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! |
− | | + | </p> |
| <figure> | | <figure> |
| <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"> |
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| </figure> | | </figure> |
| <br><br> | | <br><br> |
− | <h2>Cell cytometry to examine gene expression levels</h2> | + | <h2 class="subtitle">Cell cytometry to examine gene expression levels</h2> |
− | | + | <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 S. elongatus 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. |
| + | </p> |
| <figure> | | <figure> |
| <img src="https://static.igem.org/mediawiki/2019/2/27/T--Marburg--UDARyfpFACSmeasurement.png" alt="UTEXwtYFP"> | | <img src="https://static.igem.org/mediawiki/2019/2/27/T--Marburg--UDARyfpFACSmeasurement.png" alt="UTEXwtYFP"> |
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| </figure> | | </figure> |
| <br><br> | | <br><br> |
| + | <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 throughtout 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 throughtout the whole experiment, while at higher light intensities a shift towards higher fluorescence intensities could be observed. |
| + | </p> |
| <br><br> | | <br><br> |
| <figure> | | <figure> |
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| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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− | </p>
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| </div> | | </div> |
| </div> | | </div> |