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 ight 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.

xxxx Fig x.:Schema vom Workflow
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 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.

fig XX (screenshot des messprogams)
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 1350nm 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.
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