Part:BBa_K1065001
AraC-pBAD + 2-oxoglutarate oxygenase/decarboxylase (EFE) + terminators
This is a composite part composed by an AraC-pBAD promoter (BBa_K731201) + 2-oxoglutarate oxygenase/decarboxylase (BBa_K1065000) + terminators (BBa_B0015). We used this part to characterize the Ethylene Forming Enzyme, inducing its expression with Arabinose.
Safety
This part produces Ethylene. With an air volume/culture volume ratio = 4, we detected about 200 ppm of Ethylene. This concentration is not dangerous and not inflammable (with a concentration from 27000 to 320000 ppm, Ethylene can be inflammable). However we suggest to manage this part carefully.
EFE characterization
In order to obtain a good characterization of the part, a set of experiments were done.
Effect of EFE on cell growth
Figure 1 Effect of EFE on cell growth. Cell density was measured at different time points to determine the effect of EFE expression. Cells were grown at 37 °C in LB until it was reached an OD of 0.6. The cells were then slitted 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
Ethylene detection through Micro Gas-Chromatography
Figure 2 Ethylene detection through Micro GC. Cells were grown until O.D.600 reached 0.5. The cells were then splitted in two samples of equal volume (3 ml) and putted into an ermetically closed vial with a pierceable septum. One of the two sample was induced with 5 mM Arabinose. The vials were then putted into the thermoshaker for 4 hours. After that, the vials were connected to a micro GC and a measure was taken. Looking at the chromatograms, we can easily note that induce sample showed a characteristic peak corresponding to Ethylene. On the other hand, the not induce sample didn't show that peak (panel A). Panel B: picture of the vial connected to the micro GC.
Kinetic assay for Ethylene production
Figure 3 Kinetic assay for Ethylene production. Cells were grown as described in figure 2 whith the only difference that a stirrer was also putted into the vial. This is due to the fact that the vial had to be connected to the micro GC and stirred at 37 °C for the entire duration of the experiment. A measure was taken every 45/60 mins for about 8 hours. As you can see, samples were induced at two differents O.D.600 and this had big effect on the amount of ethylene produced. However, seems that the Ethylene concentration in the air space reached saturation level after only two hours. Moreover when the blue curve was registered, a sample of the same stock culture was kept in thermoshaker. As expected an higher value of ethylene was detected (green point) since it was subjected to only one measure (thus having no gas loss due to repeated measures).
Acceleration of fruit ripening
Figure 4 Acceleration of fruit ripening. Our system was exploited for the acceleration of fruit ripening. We designed an ermetically closed jam 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 his O.D.600 reached 0.8. The flasks contained also a stirrer.The cultures were maintained at 37 °C using a laboratory heating plate connected to a digital thermometer immersed in the culture. For four to six days, every morning the culture in the flasks was substituted with a new induced (or not) colture. Furthermore, canonical jam jars (i.e.: with no connector) were adopted to contain the negative control fruit samples (panel A). We tested many type of fruit, using different time of treatment. In this example (panel B) the tomatoe putted in the jar that was connected to an induced culture, shows a more advance stage of maturation after 4 days of threatment.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1530
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1228
Illegal AgeI site found at 979
Illegal AgeI site found at 2281 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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