Light measurement
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Revision as of 13:49, 19 October 2019
M E A S U R E M E N T
Amplifying new standards in measurement
Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
Storytelling:
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 but still standardization is a huge part in synthetic microbiology
and
necessary to compare results with other scientists and reproduce their data.
While we wanted to establish Syn. elong. 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
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 on how to cultivate UTEX at 1500 μE.
[quelle].
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
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 popup we
show
different ways of measurement, their (dis-)advantages and different results depending on the measuring
instrument.
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
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.
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 approach
was to
measure the fluorescence/luminescence with a plate reader [link part measurement]. 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) [link
facs]. In
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 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 difference in
measurement
methods.
At 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 measurement.
Light Measurement
Abstract?
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Reporters
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Reporter
Abstract?
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Fluorescence-Activated Cell Sorting (FACS)
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Fluorescence-Activated Cell Sorting (FACS)
Abstract?
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Part Measurement
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.
Part Measurement
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 Collection 2.0 with XXX 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 different cultivation vessels and parameters - and revised them after evaluating the results. In the end we were able to establish a workflow specially designed for our methods to cultivate and characterize the parts from our Marburg Collection 2.0, that is tailored to Synechococcus elongatus UTEX 2973.
The results of our part characterization were obtained by fluorescence and
luminescence
measurements (of what?). But before the party could be measured we had to elaborate
a
cultivating and measuring workflow.
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 well plates, because the Marburg
Collection 2.0 comprises xxx parts and we were limited in space in our incubator.
Starting with 96-well-plates it was impossible to cultivate Synechococcus
elongatus UTEX 2973 under our conditions (hier aufführen?) since the
cultures
showed small clouds of cells formed by inappropriate movement of media in the wells.
In
addition, the rpm of the incubator was limited whereas cultures in flasks had to be
incubated at the same time and these threatened to fall over at high rpm. At 130 rpm
we
found a compromise between cultivating flasks and well-plates in the same incubator.
After revising the workflow over and over we came to the conclusion, that it is
favorable to cultivate the UTEX 2973 in transparent 24-well-plates 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 light
conditions. Concerning of light conditions, we evaluated that the cells showed good
(prosperous?) 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 (ich glaub die flache ist eher klein, aber
vllt
wegen der Temperatur und Zeit?). Further it is essential to know the volume in the
wells
for measuring in the plate reader. Therefore we compared different seals for the
well
plates and in the end we came to the conclusion that using a semipermeable foil is
the
best solution. The evaporation could be minimalized and the cells were able to get
enough CO2 because air transfer was provide/permit. By using a foil it was possible
to
cultivate the cells for 2-3 days without losing significant amounts of medium.
As described before we used the following workflow as shown in fig. XX to cultivate and measure our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the end of the triparental conjugation (LINK). For every part we picked 3 different colonies and inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to OD730=0.6-0.8 they were inoculated to 1.0 mL of OD730=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 inoculated with 1.0 mL of a OD730= 0.1 UDAR culture that was used as a blank while evaluating the results (that will be used as a blank while ...). When all the cultures in the second 24-well-plate reached OD730=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 technical parallels 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 OD730=0.6-0.8 the cultures were transferred into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three times. Following this workflow we were able to measure three biological parallels and two technical parallels for every biological parallel. It enabled us to have a good statistical database and gives our results a stronger meaning/significance. While working with this workflow it was essential to keep the cultures in their exponential phase because it would significantly speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es erst gar keine lag phase gibt).
Concerning the measurement part we decided to transfer the cultures into black/white 96-well-plates. While fluorescence is measured in black well-plates the luminescence is 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. Further we could measure eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for measurement)
Fluorescence measurement:
After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the considered part. For measurement we created a program that measured the OD730 and the fluorescence of the wells.
In order to measure the OD in each well we determined the absorption at 730 nm. Further we measured multiple points in each well, where 3x3 points (circular) with a gap of 1350 nm to the border of the well showed consistent results with small standard deviations (fig. XX). We used the same settings of the multiple measurement for the fluorescence measurement. While using sYFP as 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 shown in XX (Database verlinken/als quelle?)
Fluorescence-Activated Cell Sorting (FACS):
short abstract and link to the FACS-text of the measurement
Luminescence Measurement
text
kein plan was man hier schreiben soll zum jetzigen standpunkt...
For analyzing the data we used two blanks. For OD measurement we used pure medium
(BG11)
and for the fluorescence measurement we used UTEX 2973 without a fluorescent
protein.
Auswertung, Daten und Grafen darstellen?
Growth Curves
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Growth Curves
Abstract?
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