Difference between revisions of "Team:Marburg/Measurement"

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               Fluorescence Activated Cell Sorting (FACS) is a flow cytometry measurement technique that separates single cells with different fluorescence characteristics. In this method you get accurate measuring results, because every single cell is analyzed on its own. FACS analysis can be used for cell sorting, fluorescence analysis of single cells and cell counting of different sample mixtures.
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               Fluorescence Activated Cell Sorting (FACS) is a flow cytometry measurement technique that separates single cells with different fluorescence characteristics. In this method one gets accurate measuring results, because every single cell is individually analyzed. FACS analysis can be used for cell sorting, fluorescence analysis of single cells and cell counting of different sample mixtures.
 
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               In flow cytometry, the sample with the cells get hydrodynamically focused in a single stream. The cells arrange in a row, so that they can be illuminated with a laser one by one. The cells get excited by the laser and emit light at various wavelengths, which is then detected by a photon detector. The detector can detect and separate different fluorescence intensities at a definite range of wavelengths. Through this, cells can be categorized via different fluorescent characteristics and, if needed, separated into defined categories by a deflection system using electromagnetic fields.  
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               In flow cytometry, the sample with the cells gets hydrodynamically focused in a single stream. The cells arrange in a row, so that they can be illuminated with a laser one by one. The cells get excited by the laser and emit light at various wavelengths, which is then detected by a photon detector. The detector can detect and separate different fluorescence intensities at a definite range of wavelengths. Through this, cells can be categorized via different fluorescent characteristics and, if needed, separated into defined categories by a deflection system using electromagnetic fields.  
 
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               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 probe 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>
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               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 probe 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>
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               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>
 
               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, one of them being the one of the beads and the other one from 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, one of them being the one of the beads and the other one from 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:
 
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Revision as of 13:33, 8 December 2019

M E A S U R E M E N T


Amplifying new standards in measurement

We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 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 cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore it is hard to hold on to special methods; nevertheless, standardization is paramount in synthetic microbiology in order to be able to compare results with other scientists and reproduce their data.

Because we wanted to establish Synechococcus elongatus as a new chassis for the iGEM community and scientists, we should find 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 light measurement, evaluated different reporters, established a measurement method and compared it to a already known FACS measurement method.

At the beginning of our project we faced the first question: how to cultivate UTEX at 1500 μE? To answer this 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 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 popups we show different ways of measurement, their (dis-)advantages and different results depending on the measuring instrument.

Moreover, 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, we recognized that small deviations of these 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 that lead to the fastest growth speed.

Other aspects were measuring the expression and characterizing our part. Different possibilities were discussed and after testing them we decided on two methods in our project. One 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. 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 own.

However, not every laboratory possesses a FACS device. So in the end we would like to offer a database - analyzed using these two methods - of our constructs for iGEM teams and research groups, who do not have access to a FACS device and show the difference in measurement methods.

In the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 and make a contribution to the cyano community by establishing essential/fixed standards in measurements.


L I G H T
M E A S U R E M E N T


Light measurements are a crucial aspect when working on phototrophic organisms - here’s how we tackled some issues we faced!

R E P O R T E R S


Fluorescence Reporters

F A C S


FACS Measurements

P A R T
M E A S U R E M E N T


Establishing a measurement workflow that is not only applicable to UTEX 2973 and other cyanobacteria with a high throughput.

G R O W T H
C U R V E S


Varying our growth conditions we were finally able to achieve doubling times of under 80 minutes.