Part:BBa_K4624626

PFR1-syfp2-rrnB T1/T7TE + AraC/PBAD-fapR-rrnB T1/T7TE

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2064
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1899
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1881
  

Introduction

This construct was designed by iGEM Thessaly 2023 team, to evaluate the repression that the regulatory protein FapR (BBa_K4624003) poses on the synthetic promoter P_FR1 (BBa_K4624000).

Experimental Design and Results

The GoldneBraid 2.0 cloning method was used, which means that each part contained in this construct, had to initially be cloned into the universal part domesticatoion vector pUPD2. All the necessary seqences were 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. After numerous trials, the final compostite part was succesfully asembed, confirmed with a diagnostic digestion (Fig. 1).

Figure 1: 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).

For this experiment, we started by transforming E. coli BL21 (DE3) chemically competent cells with the isolated plasmid carrying the level 2 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 positive (syfp2 under the regulation of the Anderson J23118 promoter) and negative control (non-transformed cells). 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. The final results are depicted in Fig. 2.

Figure 2: 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. 2), 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.