Part:BBa_K4624625

FadBA promoter

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_fadBA is a fatty acyl-CoA-responsive promoter derived from the Escherichia coli fadBA operon, which encodes the fatty-acid-oxidizing multienzyme complex ^[1]. P_fadBA contains binding site for FadR (BBa_K4624005), a transcription factor regulated by acyl-CoA thioestres ^[2]. In the absence of fatty acids, FadR is expected to bind to the specific 17-bp recognition site, interfering with RNA polymerase. When fatty acids are present, they are activated to acyl-CoAs by acyl-CoA synthase (encoded by fadD). Acyl-CoAs in turn bind to FadR and release the protein from the promoter.

Experimental Design and Results

Our team decided to attempt a basic characterization, by firstly designing a construct (Fig. 1) with the P_fadBA upstream of a reporter gene encoding the SYFP2 (BBa_K864100), a monomeric fluorescent protein with optimized folding, maturation and a narrow fluorescence emission spectrum6, contained in this year’s Distribution kit.

Figure 1: Schematic representation of the level 1 (alpha) construct we designed for the characterization of the P_fadBA .

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 units. The sequence of P_fadBA 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. 2), we successfully constructed the level 0 construct.

Figure 2: Diagnostic digestion of pUPD2_pfadBA with EcoRI and EcoRV, expected bands (bp): 1309 and 896. Lane 2: 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. 3).

Figure 3: Diagnostic digestion of pDGB3α1_pfadBA-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): 2327, 2026, 1628 and 1291. Lane 2: pDGB3α1 (no insert).

To adequately characterize P_fadBA, 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] (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.4).

Figure 4: Normalized fluorescence intensity measurement for pfadBA-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. Yang XY, Schulz H, Elzinga M, Yang SY. Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli. Biochemistry. 1991 Jul 9;30(27):6788-95. doi: 10.1021/bi00241a023. PMID: 1712230.</p>

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.