Part:BBa_K4624010

PFR1-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]

Intoduction

This level 1 (alpha) construct carrying the P_FR1 upstream the reporter syfp2 was designed to evaluate the strenght of the promoter, a new basic part added by our team. By assembling this construct and comparing its fluorescence output to that of a standard promoter (P_J23118) we aimed to provide future users with a straightforward way to assess the suitability of P_FR1 for their specific purposes.

Experiments

For the assembly of this construct, we utilized the GoldenBraid 2.0 cloning method. Initially, the sequence of the promoter was domesticatd, a process involving the removal of internal restriction sites that were not compatible with the GoldenBraid standards and the addition of appropriate 3' and 5' 4-nt overhangs. Once we acquired the domesticated sequence, the next step was to proceed with the assembly.

The domesticated sequence was inserted into the pUPD2 part domestication vector (BBa_K3505007) to create the level 0 construct. Once the insertion was verified through diagnostic restriction-digestion reaction (Fig. 1), we used the resulting level 0 construct along with the constructs of the B0030 RBS (BBa_J428032), syfp2 (BBa_K864100) and B0015 double terminator (BBa_J428092), provided by this year’s iGEM distribution kit, to create the level 1 reporter construct. The assembly was succesful and the insert was verified through diagnostic restriction-digestion reaction (Fig. 2).

Figure 1: Diagnostic digestion of pUPD2_pFR1 with EcoRI and EcoRV, expected bands (bp): 1305 and 896. Lane 2: pUPD2 (no insert).

Figure 2: Diagnostic digestion of pDGB3α1_pFR1-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): 2327, 2022, 1628, 1291 and 58. Lane 2: pDGB3α1 (no insert).

1st experiment

Having the reporter construct (Fig. 2) ready, we set out to perform the experiment for the evaluation of the promoter. In short, the isolated plasmid carrying the reporter construct was used to transform E. coli BL21 (DE3) chemically competent cells, following standard protocol. Single colonies were picked to inoculate 5 ml of LB medium, with the appropriate antibiotic, and cultured O/N at 37^οC and 210 rpm. Single colonies were also picked for the positive control (syfp2 under the regulation of the Anderson J23118 promoter (BBa_K4624621), and the negative control (non-transformed E. coli BL21 DE3 cells). The O/N cultures were used to prepare the final dilutions into M9 minimal medium in order to reach the same starting OD₆₀₀. Measurements were taken after 6 hours of incubation (37^οC and 210 rpm) at 511 nm (excitation) and 529 nm (emission) (Fig. 3).

Figure 3: Normalized fluorescence intensity of the pFR1-syfp-rrnB T1/T7TE construct, compared to a positive control (pJ23118-syfp-rrnB T1/T7TE) and a negative control (non-transformed E. coli cells) after 6h incubation.

It is evident that the P_FR1 exhibits a fluorescence output similar to that of the constitutive P_J23118, in absence of the FapR regulatory protein. So we can conclude that these two promoters have a comparable intensity.