Part:BBa_K4624601

AR synthetic promoter (PAR)

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]

Biology

P_AR is a synthetic fatty acyl-CoA-responsive promoter designed based on a phage T7 promoter (P_A1) ^[1]. P_AR contains FadR-recognition sites from P_fadBA (BBa_K4624625), which increases its dynamic range, making it a suitable promoter for the construction of a fatty acyl-CoA biosensor ^[2].

Experimental design and results

Our team used this promoter 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 its intensity 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.

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_AR 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 (2) pUPD2_pAR with EcoRI annd EcoRV, expected bands (bp):(2) 1305 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 that we could then test. We successfully built the level 1 (alpha) construct, which was confirmed with a restriction digestion reaction (Fig. 2).

Figure 2: Diagnostic digestion of (2) pDGB3α1_pAR-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): (2) 2327, 2022, 1628, 1291 and 58. Lane 3: pDGB3α1 (no insert).

To adequately characterize P_AR, we included two controls: 1. a positive control construct which we designed and built, carrying the same reporter gene (syfp2) under the control of the well-documented constitutive Anderson promoter J23118, 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 ^[3] (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 were taken after 6h incubation. The results we got are shown below (Fig.3).

Figure 3: Normalized fluorescence intensity measurement for pAR-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.

References

1. Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. doi: 10.1093/nar/25.6.1203. PMID: 9092630; PMCID: PMC146584.

2. Zhang F, Carothers JM, Keasling JD. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol. 2012 Mar 25;30(4):354-9. doi: 10.1038/nbt.2149. PMID: 22446695.

3. Römer A, Rawat D, Linn T, Petry SF. Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity. Biol Methods Protoc. 2021 Dec 3;7(1):bpab023. doi: 10.1093/biomethods/bpab023. PMID: 35036572; PMCID: PMC8754478.