Light measurement
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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.
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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.
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.
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?
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