Part:BBa_K4624624

PLR-syfp2-rrnB T1/T7TE

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Engineering Cycle 1 - Regulation of the phaJ gene

Our team used this construct as part of a DBTL cycle aimed at optimizing the regulation of the phaJ gene we have implemented on our Design. Our objective was to evaluate the intensity of the fatty acyl-CoA-responsive promoter P_LR (BBa_K4624602) building a construct of the promoter regulating the expression of the reporter gene syfp2 (BBa_K864100), and compare its output to positive control, the J23118 Anderson promoter expressing the same reporter gene (BBa_K4624621).

To assemble this construct with the GoldenBraid 2.0 cloning method, we first had to insert the promoter into a universal part domestication vector such as the pUPD2, in order to create a level 0 construct which could then be combined with other level 0 constructs to assemble a complete transcription unit. The sequence of P_LR was acquired and then domesticated, using the GoldenBraid Domesticator tool, which removes any internal restriction sites that did not comply with the GoldenBraid standards and adds the appropriate 4-nt 3’ and 5’ flanking overhangs in order for the inserts to be compatible with our level 0 pUPD2 cloning vector.

Through digestion-ligation reaction, DH5α chemically competent cells transformation, plasmid isolation and restriction-digestion confirmation (Fig. 1), we successfully constructed the level 0 construct.

Figure 1: Diagnostic digestion of (1) pUPD2_pLR with EcoRI and EcoRV, expected bands (bp): (1) 1300 and 896. Lane 3: pUPD2 (no insert).

The level 0 construct was then combined, via a digestion-ligation reaction, with the Distribution kit’s level 0 constructs of the B0030 RBS (BBa_J428032), the syfp2 (BBa_K864100) and the B0015 double terminator (BBa_J428092), in order to build the complete transcriptional unit. We successfully built the level 1 (alpha) construct, which was confirmed with a restriction-digestion reaction (Fig. 2).

Figure 2: Diagnostic digestion of (1) pDGB3α1_pLR-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): (1) 2327, 2022, 1628, 1291 and 58. Lane 3: pDGB3α1 (no insert).

To adequately characterize P_LR, we included two controls: 1. a construct carrying the same reporter gene (syfp2) under the control of the well-documented constitutive Anderson promoter J23118, 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 (for 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. 3).

Figure 3: Normalized fluorescence intensity measurement for pLR-syfp-rrnB T1/T7TE construct on three different conditions (No oleic acid addition, addition of non-dissolved oleic acid, addition of oleic acid dissolved in DMSO) after 6h incubation.