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). This construct allowed us to characterize the Ethylene Forming Enzyme, inducing its expression using Arabinose at a concentration of 5 mM.

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. Afer 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 (panel A). On the other hand, the not induce sample didn't show that peak (panel B). Panel C: picture of the vial connected to the micro GC.

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