Part:BBa_K4624003

FapR transcription factor from Bacillus subtilis

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]

Introduction

FapR is a malonyl-CoA-responsive transcription factor derived from the Gram-positive bacteria Bacillus subtilis, which specifically binds to a 17-bp DNA sequence and negatively regulates the expression of many genes involved in fatty acid and phospholipid metabolism ^[1]. The binding of malonyl-CoA to FapR triggers a conformational change to the protein, causing the dissociation of the FapR−DNA complex ^[2].

Experimental Design and Results

Our team managed to conduct an initial part characterization and test the functionality of the FapR along with the P_FR1 (BBa_K4624000), a synthetic promoter with FapR-binding sites, for the construction of a malonyl-CoA based negative feedback system ^[3].

To evaluate the levels of represion the protein exerts on the P_FR1, a composite part was designed, containing a reporter construct of the P_FR1 regulating the expression of the reporter gene syfp2 (BBa_K864100) and an additional one, carrying the fapR regulated by the AraC/P_BAD system (Fig. 1). So, by titrating arabinose to control the levels of FapR we will be able to determine the levels of repression the protein exerts on the promoter, and the degree of fapR transcription required to make the system responsive.

Figure 1: Schematic representation of the level 2 (omega) construct for the evaluation of the inhibition FapR poses on P_FR1.

To assemble this construct with the GoldenBraid 2.0 cloning method, we first had to clone fapR 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 was acquired and then domesticated, using the GoldenBraid Domesticator tool , which removes any internal restriction sites that did not comply with the GoldenBraid standard 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.

When this first step was succesfully completed (Fig. 2), another digestion-ligation reaction was performed to combine fapR with the AraC/P_BAD composite part and the B0015 double terminator (BBa_J428092).

Through DH5α chemically competent cells transformation, plasmid isolation and restriction-digestion confirmation, we successfully assembled the 1st transcription unit (Fig. 3).

Figure 2: Diagnostic digestion of pUPD2_fapR with EcoRV, expected bands (bp): 1477 and 1196. Lane 2: pUPD2 (no insert).

Figure 3: Diagnostic digestion of pDGB3α2_araC/pBAD-fapR-rrnB T1/T7TE with EcoRV and BsaHI, expected bands (bp): 2327, 1628, 1379, 1291, 807, 559, 391 and 58. Lane 2: pDGB3α2 (no insert).

This level 1 (alpha) construct was then combined with the P_FR1-driven reporter module to create the construct for the final characterization (Fig. 1). Through another digestion-ligation reaction, plasmid isolation and restriction-digestion confirmation, we successfully assembled the desired composite part (Fig. 4).

Figure 4: Diagnostic digestion of pDGB3ω1_pFR1-syfp-rrnB T1/T7TE + araC/pBAD-fapR-rrnB T1/T7TE with EcoRI and BsaHI, expected bands (bp): 2325, 1898, 1628, 1304, 1291, 758, 470 and 58. Lane 2: pDGB3ω1 (no insert).

Now it was time to test our construct. For experiment, we started again by transforming E. coli BL21 (DE3) chemically competent cells with the isolated plasmid carrying the construct. The next day, for the preparation of liquid cultures, single colonies were picked and inoculated in LB medium, with the appropriate antibiotic, and finally the cultures were incubated O/N at 37^οC and 210 rpm. The next morning, final dilutions x5 were prepared in M9 minimal medium for the level 2 construct as well as for the same positive and negative controls used in the 1st experiment. Lastly, addition of L-arabinose followed for the preparation of 4 different final concentrations (0.01, 0.1, 1 and 10 mM), creating various levels of P_BAD induction. Measurements at wavelengths of 511 nm (excitation) and 529 nm (emission) were taken at 3h and 6h timepoints.

Figure 5: Normalized fluorescence intensity for the level 2 construct (pDGB3ω1_pFR1-syfp-rrnB T1/T7TE + araC/pBAD-fapR-rrnB T1/T7TE) in different concentrations of L-arabinose, after 3h and 6h incubation.

As we can see from the results (Fig. 5), there is a significant decrease of fluorescence intensity at increasing concentrations of L-arabinose. This fact confirms the existence of the FapR-mediated P_FR1 repression, since higher concentration of L-arabinose implies a higher activation of the P_BAD, through the AraC regulatory protein, and ultimately a higher expression of fapR.

References

1. Schujman GE, Paoletti L, Grossman AD, de Mendoza D. FapR, a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis. Dev Cell. 2003 May;4(5):663-72. doi: 10.1016/s1534-5807(03)00123-0. PMID: 12737802.

2. Schujman GE, Guerin M, Buschiazzo A, Schaeffer F, Llarrull LI, Reh G, Vila AJ, Alzari PM, de Mendoza D. Structural basis of lipid biosynthesis regulation in Gram-positive bacteria. EMBO J. 2006 Sep 6;25(17):4074-83. doi: 10.1038/sj.emboj.7601284. Epub 2006 Aug 24. PMID: 16932747; PMCID: PMC1560364.

3. Liu D, Xiao Y, Evans BS, Zhang F. Negative feedback regulation of fatty acid production based on a malonyl-CoA sensor-actuator. ACS Synth Biol. 2015 Feb 20;4(2):132-40. doi: 10.1021/sb400158w. Epub 2014 Jan 10. PMID: 24377365.