Difference between revisions of "Part:BBa K1065000"

Figure 1 Effect of EFE on cell growth. Cell density was measured at different time points to determine the effect of EFE expression. Neb10β cells were grown at 37 °C in LB until it was reached an OD of 0.6. The cells were then splitted in four samples of equal volume. Two samples were then induced with 5 mM Arabinose. Induced samples show a slowed growth rate, as espected (5mM arabinose is a strong induction that causes stress on cells). However, cell growth is not completely inhibited so EFE is not highly toxic

Figure 2 Ethylene detection with an Agilent 3000 Micro GC set up with a plot U column. Neb10β cells were grown until O.D.600 reached 0.5. The culture was then splitted in two samples of equal volume (3 ml) and placed into an hermetically closed vial with a septum with a rubber cap. One of the two sample was induced with 5 mM Arabinose. The vials were left in the thermoshaker for 4 hours. After that, the vials were connected to a micro GC and a measure was taken. Panel A: a 1.5 ml sample induced (green curve) and 3 ml sample induced (red curve) showed a characteristic peak corresponding to Ethylene. On the other hand, the 3 ml not induced sample (blue curve) didn't show the peak. Ethylene was estimated to be 61 ± 15 ppm for the 1.5 ml culture and 101 ± 15 ppm for the 3 ml culture. Panel B: picture of the vial connected to the micro GC.

Figure 3 Kinetic assay for Ethylene production. Neb10β cells were grown until an O.D. of 0.5 - 0.8 and then connected to the micro GC, while in agitation on a thermoblock at 37 °C for the entire duration of the experiment. Every 45/60 mins a meausure was taken for a total of about 8 hours. Samples were induced at two differents O.D.600 and this had big effect on the amount of ethylene produced. However, it seems that the Ethylene concentration in the air space reached saturation after only two hours. The red dashed line indicates the amount of ethylene detected with a culture left in the thermoshaker for the all duration of the experiment and subjected to only one measurement. As expected an higher value of ethylene was measured due to the minimal gas loss with this approach.

Figure 4 Acceleration of fruit ripening. Panel A: our system was exploited for the acceleration of fruit ripening. We designed an hermetically closed jar with a rubber hose connector. These jars contained our test-fruit and each one was connected to a flask. The flasks contained 300 ml of induced (or not) culture when O.D.600 reached 0.8. The flasks contained a cultures maintained at 37 °C using a laboratory heating plate while stirring. For four to six days, every morning the culture in the flasks was substituted with a new induced (or not) culture. Furthermore, non-modified jars (i.e.: with no connector) were adopted to contain the negative control fruit samples (no-cells). Panel B: ripening of plums. Plum exposed to ethylene, show a more advanced stage of ripening after 4 days of treatment when compared to two negative controls (no-cells and BBa_K1065001 not induced).

Figure 5 Neb10β cells transformed with BBa_K1065311 were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure (92 ± 15 ppm), while it is not produced in the dark.

Figure 6 Neb10β cells transformed with BBa_1065309 were grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (37 ± 15 ppm) instead in the presence of blue light there was no ethylene produced.