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| best growing conditions. Due to the size of the Marburg Collection 2.0 (55 parts) and space limitation | | best growing conditions. Due to the size of the Marburg Collection 2.0 (55 parts) and space limitation |
| in our incubator, our first thought was using well-plates. We started with 96-well-plates and found out that it was impossible to cultivate <i>Synechococcus | | in our incubator, our first thought was using well-plates. We started with 96-well-plates and found out that it was impossible to cultivate <i>Synechococcus |
− | elongatus</i> UTEX 2973 in incubator with 130 rpm. The rpm of | + | elongatus</i> UTEX 2973 in an incubator with 130 rpm. The rpm of |
| the incubator was limited because cultures in flasks had to be incubated at the same time, presenting risks of | | the incubator was limited because cultures in flasks had to be incubated at the same time, presenting risks of |
| falling over at higher rpm. After revising the workflow over and over we came to | | falling over at higher rpm. After revising the workflow over and over we came to |
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| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
| | | |
− | Additionally, it was necessary to use transparent wells to ensure every well with similar light | + | Additionally, it was necessary to use transparent wells to ensure every well would be provided with similar light |
| conditions. Concerning the light conditions, we evaluated that the cells showed good | | conditions. Concerning the light conditions, we evaluated that the cells showed good |
| growth in the wells at low-light conditions (around 500 µE). The evaporation of medium played an | | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium played an |
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| semipermeable foil is the best solution. The evaporation could be minimalized and the cells were | | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were |
| able to get enough CO2 thanks to improvement in air circulation. By using a foil it was possible to | | able to get enough CO2 thanks to improvement in air circulation. By using a foil it was possible to |
− | cultivate the cells for 2-3 days without losing significant amount of media. | + | cultivate the cells for 2-3 days without losing significant amounts of media. |
| <br> | | <br> |
| <br> | | <br> |
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| <p> | | <p> |
| <br> | | <br> |
− | As described before we used the following workflow as shown in Fig. 1 to cultivate and measure | + | As described before we used the workflow as shown in Fig. 1 to cultivate and measure |
− | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the | + | our parts. The cultivation started by picking colonies from BG11-agar-plates that were transformed via triparental conjugation. For every part we picked three different colonies and |
− | end of the triparental conjugation. For every part we picked three different colonies and
| + | |
| inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates | | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
− | we could inoculate eight different parts with three biological parallels. When the cultures grew to | + | we could inoculate eight different parts with three biological replicates. When the cultures grew to |
| OD<sub>730</sub>=0.6-0.8 they were inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells | | OD<sub>730</sub>=0.6-0.8 they were inoculated to 1.0 mL of OD<sub>730</sub>=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 | | 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 OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while | + | inoculated with 1.0 mL of an OD<sub>730</sub>= 0.1 untransformed culture that was used as a blank while |
| evaluating the results. When all the cultures in the | | evaluating the results. When all the cultures in the |
| second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same | | second 24-well-plate reached OD<sub>730</sub>=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 | | 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 | + | technical replicates 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 OD<sub>730</sub>=0.6-0.8 the cultures were transferred | | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
| into a 96-well-plate. Every well of the 24-well-plate was measured three | | into a 96-well-plate. Every well of the 24-well-plate was measured three |
− | times. Following this workflow we were able to measure three biological parallels and | + | times. Following this workflow we were able to measure three biological replicates and |
− | two technical parallels for every biological parallel. It enabled us to have a good statistical | + | two technical replicates for every biological replicate. It enabled us to have a good statistical |
| database and gives our results a stronger meaning and significance. While working with this workflow | | database and gives our results a stronger meaning and significance. While working with this workflow |
| it was essential to keep the cultures in their exponential phase because it would significantly | | it was essential to keep the cultures in their exponential phase because it would significantly |
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| <p style="text-align: justify; margin-bottom: 1em;"> | | <p style="text-align: justify; margin-bottom: 1em;"> |
| | | |
− | Concerning the measurement part we decided to transfer the cultures into black/white luminescence | + | For the measurement we decided to transfer the cultures into black/white well plates and luminescence |
− | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every | + | was 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. Furthermore we could measure | | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Furthermore we could measure |
| eight parts simultaneously in one plate. (four 24-well-plates lead into one 96-well-plate for | | eight parts simultaneously in one plate. (four 24-well-plates lead into one 96-well-plate for |
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| measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the | | 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. We used the | | border of the well showed consistent results with small standard deviations. We used the |
− | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as | + | same settings for all fluorescence measurements. While using sYFP as a |
| signal for our part measurement we have set the emission wavelength to 515 nm and the excitation | | 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.<br> | + | wavelength to 527 nm, fitting the exact wavelengths of sYFP.<br> |
| <br> | | <br> |
| </p> | | </p> |