Part:BBa_K1065001

AraC-pBAD + 2-oxoglutarate oxygenase/decarboxylase (EFE) + terminators

This device is 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, a compound that can be inflammable at a concentration between 2.7 to 36%. We characterized this part under the control of an AraC-pBAD promoter. With a air volume/culture volume ratio = 4, we detected about 200 ppm of Ethylene. This concentration is not dangerous and not inflammable. However we suggest to manage this part carefully.

EFE characterization in Neb10bβ cells

Figure 1 Effect of EFE on cell growth. Cell density was measured at different time points to determine the effect of EFE expression. Neb10bβ cells were grown at 37 °C in LB until it was reached an OD of 0.6. The cultures 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

Ethylene detection through Micro Gas-Chromatography

Figure 2 Ethylene detection with an Agilent 3000 Micro GC set up with a plot U column. Neb10bβ cells were grown until O.D.600 reached 0.5. The culture was then splitted in two samples of equal volume (3 ml) and putted 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.

Kinetic assay for Ethylene production

Figure 3 Kinetic assay for Ethylene production. Neb10bβ 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.

Acceleration of fruit ripening

This device was used for accelerating fruit ripening. Many type of fruit were tested and ripened successfully. For more information and details please visit the UNITN wiki page .

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).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1530
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1228
  Illegal AgeI site found at 979
  Illegal AgeI site found at 2281
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961