**Engineering Cycle 1 - Regulation of the phaJ gene**

To evaluate the suitability of the fatty acyl-CoA promoter P_fadBA (BBa_K4624625) in our Design, we built a construct carrying the promoter upstream of the syfp2 (BBa_K864100), in order to measure the fluorescence intensity output. The successful insertion of this construct into the pDGB3α1 vector was confirmed with a restriction-digestion reaction and gel electrophoresis (Fig. 1).

To adequately characterize P_fadBA, we included two controls: 1. a construct carrying the same reporter gene (syfp2) under the control of the well-documented constitutive Anderson promoter J23118 (BBa_K4624621), as a positive control and 2. non-transformed BL21 (DE3) cells as a negative control. Single colonies were picked for both constructs, as well as a colony of non-transformed BL21 (DE3) cells and inoculated into 5 ml of LB with the appropriate antibiotic. The cultures were grown O/N at 37^oC and 210 rpm. The next day, a dilution was performed to reach an OD₆₀₀ of approximately 0.02 and a black plate with a transparent bottom was prepared, blanks included. The construct was tested in three different conditions: with the addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO (final concentration 10mM in each case), and without oleic acid. Four biological repeats were performed for each condition, the plate was placed into the plate reader and measurements of absorbance and fluorescence intensity were taken after 6h incubation. The results we got are shown below (Fig.2).

Part:BBa_K4624622

Engineering Cycle 1 - Regulation of the phaJ gene

**Engineering Cycle 1 - Regulation of the phaJ gene**